RAS Mediates BET Inhibitor-Endued Repression of Lymphoma Migration and Prognosticates a Novel Proteomics-Based Subgroup of DLBCL through Its Negative Regulator IQGAP3

Phenotypic heterogeneity and molecular diversity make diffuse large B-cell lymphoma (DLBCL) a challenging disease. We recently illustrated that amoeboid movement plays an indispensable role in DLBCL dissemination and inadvertently identified that the inhibitor of bromodomain and extra-terminal (BET) proteins JQ1 could repress DLBCL migration. To explore further, we dissected the impacts of BET inhibition in DLBCL. We found that JQ1 abrogated amoeboid movement of DLBCL cells through both restraining RAS signaling and suppressing MYC-mediated RhoA activity. We also demonstrated that BET inhibition resulted in the upregulation of a GTPase regulatory protein, the IQ motif containing GTPase activating protein 3 (IQGAP3). IQGAP3 similarly exhibited an inhibitory effect on RAS activity in DLBCL cells. Through barcoded mRNA/protein profiling in clinical samples, we identified a specific subgroup of DLBCL tumors with enhanced phosphatidylinositol-3-kinase (PI3K) activity, which led to an inferior survival in these patients. Strikingly, a lower IQGAP3 expression level further portended those with PI3K-activated DLBCL a very dismal outcome. The inhibition of BET and PI3K signaling activity led to effective suppression of DLBCL dissemination in vivo. Our study provides an important insight into the ongoing efforts of targeting BET proteins as a therapeutic approach for DLBCL.

Pannexin 1 Regulates Skeletal Muscle Regeneration by Promoting Bleb-Based Myoblast Migration and Fusion Through a Novel Lipid Based Signaling Mechanism

Adult skeletal muscle has robust regenerative capabilities due to the presence of a resident stem cell population called satellite cells. Muscle injury leads to these normally quiescent cells becoming molecularly and metabolically activated and embarking on a program of proliferation, migration, differentiation, and fusion culminating in the repair of damaged tissue. These processes are highly coordinated by paracrine signaling events that drive cytoskeletal rearrangement and cell-cell communication. Pannexins are a family of transmembrane channel proteins that mediate paracrine signaling by ATP release. It is known that Pannexin1 (Panx1) is expressed in skeletal muscle, however, the role of Panx1 during skeletal muscle development and regeneration remains poorly understood. Here we show that Panx1 is expressed on the surface of myoblasts and its expression is rapidly increased upon induction of differentiation and that Panx1–/– mice exhibit impaired muscle regeneration after injury. Panx1–/– myoblasts activate the myogenic differentiation program normally, but display marked deficits in migration and fusion. Mechanistically, we show that Panx1 activates P2 class purinergic receptors, which in turn mediate a lipid signaling cascade in myoblasts. This signaling induces bleb-driven amoeboid movement that in turn supports myoblast migration and fusion. Finally, we show that Panx1 is involved in the regulation of cell-matrix interaction through the induction of ADAMTS (Disintegrin-like and Metalloprotease domain with Thrombospondin-type 5) proteins that help remodel the extracellular matrix. These studies reveal a novel role for lipid-based signaling pathways activated by Panx1 in the coordination of myoblast activities essential for skeletal muscle regeneration.

ShcD Binds DOCK4, Promotes Ameboid Motility and Metastasis Dissemination, Predicting Poor Prognosis in Melanoma

Metastases are the primary cause of cancer-related deaths. The underlying molecular and biological mechanisms remain, however, elusive, thus preventing the design of specific therapies. In melanomas, the metastatic process is influenced by the acquisition of metastasis-associated mutational and epigenetic traits and the activation of metastatic-specific signaling pathways in the primary melanoma. In the current study, we investigated the role of an adaptor protein of the Shc family (ShcD) in the acquisition of metastatic properties by melanoma cells, exploiting our cohort of patient-derived xenografts (PDXs). We provide evidence that the depletion of ShcD expression increases a spread cell shape and the capability of melanoma cells to attach to the extracellular matrix while its overexpression switches their morphology from elongated to rounded on 3D matrices, enhances cells’ invasive phenotype, as observed on collagen gel, and favors metastasis formation in vivo. ShcD overexpression sustains amoeboid movement in melanoma cells, by suppressing the Rac1 signaling pathway through the confinement of DOCK4 in the cytoplasm. Inactivation of the ShcD signaling pathway makes melanoma cells more sensitive to therapeutic treatments. Consistently, ShcD expression predicts poor outcome in a cohort of 183 primary melanoma patients.

Symmetry Breaking during Cell Movement in the Context of Excitability, Kinetic Fine-Tuning and Memory of Pseudopod Formation

The path of moving eukaryotic cells depends on the kinetics and direction of extending pseudopods. Amoeboid cells constantly change their shape with pseudopods extending in different directions. Detailed analysis has revealed that time, place and direction of pseudopod extension are not random, but highly ordered with strong prevalence for only one extending pseudopod, with defined life-times, and with reoccurring events in time and space indicative of memory. Important components are Ras activation and the formation of branched F-actin in the extending pseudopod and inhibition of pseudopod formation in the contractile cortex of parallel F-actin/myosin. In biology, order very often comes with symmetry. In this essay, I discuss cell movement and the dynamics of pseudopod extension from the perspective of symmetry and symmetry changes of Ras activation and the formation of branched F-actin in the extending pseudopod. Combining symmetry of Ras activation with kinetics and memory of pseudopod extension results in a refined model of amoeboid movement that appears to be largely conserved in the fast moving Dictyostelium and neutrophils, the slow moving mesenchymal stem cells and the fungus B.d. chytrid.

Calpain-2 regulates hypoxia/HIF-induced amoeboid reprogramming and metastasis

SummaryHypoxia, through hypoxia inducible factor (HIF), drives cancer cell invasion and metastatic progression in various cancer types, leading to poor prognosis. In epithelial cancer, hypoxia further induces the transition to amoeboid cancer cell dissemination, yet the molecular mechanisms, relevance for metastasis, and effective interventions to combat hypoxia-induced amoeboid reprogramming remain unclear. Here, we identify calpain-2 as key regulator and anti-metastasis target of hypoxia-induced transition from collective to amoeboid dissemination of breast and head and neck (HN) carcinoma cells. Hypoxia-induced amoeboid dissemination occurred through low ECM-adhesive, bleb-based amoeboid movement, which effectively invaded into 3D collagen with low-oxidative and -glycolytic energy metabolism, revealing an microenvironmentally-induced, energy-conserving dissemination route in epithelial cancers. Hypoxia-induced calpain-2 mediated amoeboid conversion by de-activating beta1 integrins, through enzymatic cleavage of the focal adhesion adaptor protein talin-1. Consequently, targeted downregulation of calpain-2 or pharmacological intervention restored talin-1 integrity, beta1 integrin engagement and reverted blebbing-amoeboid to elongated phenotypes under hypoxia. Calpain-2 activity was required for hypoxia-induced blebbing-amoeboid conversion in the orthotopic mouse dermis, and upregulated in invasive HN tumor xenografts in vivo, and attenuation of calpain activity prevented hypoxia-induced metastasis to the lungs. This identifies the calpain-2/talin-1/beta1 integrin axis as mechanosignaling program and promising intervention target of plasticity of cancer cell invasion and metastasis formation in epithelial cancers under hypoxia.

Intravital Imaging of Bright Cyan-Fluorescent AML Model Reveals Impact of CXCR4 Inhibitor on AML and Immune Cell Dynamics

INTRODUCTION: CXCR4 chemokine receptor inhibitors such as BL-8040 (BioLineRx) have been investigated by us and others as possible anti-leukemic drugs due to their ability to "mobilize" leukemia cells out of the BM and into the circulation, where they are more sensitive to chemotherapy. However, the exact mode of cell relocation remains unclear. CXCR4/CXCL12 signaling pathway also participates in BM homing of immune cells, including both central memory T cells and immunosuppressive CD4+FoxP3+ T-regulatory cells (T-reg). Therefore, CXCR4 inhibition has the potential to either counteract or enhance the process of AML immune surveillance. Therefore, we sought to develop a syngeneic AML model for intravital 2-photon microscopy (TPM) compatible with existing immune reporter mouse strains, which typically occupy the green, yellow and red fluorescence channels. HYPOTHESIS: CXCR4 inhibition decreases AML and T cell BM cellularity by increasing the rate of intravascular cell entry and/or decreasing the rate of circulating cell homing back to BM. MODEL: The cyan-colored fluorescent protein mTurquoise2 was lentivirally introduced into C57BL6-origin AML cells containing the MLL, ENL-FLT3, ITD, and p53-/- mutations, termed AML1-mTurq2. Syngeneic FoxP3-GFP/CD11c-YFP/hCD2-DsRed reporter mice were generated by inter-breeding of the corresponding strains, respectively highlighting T-reg, myeloid antigen presenting cells, and all T cells. After intravenous infusion of 1E5 AML1-mTurq2 cells, 1-2% blasts appeared in peripheral blood on day 9, increasing to 70% on day 15-20 when animals had to be euthanized. TREATMENT: Mice with >1% blasts were given BL-8040 I.P. in two daily 400 µg doses followed by imaging 24 h later, or intravenously during imaging 10 µg and 50 µg one hour later. ANALYSIS: Disease progression was characterized by blood flow cytometry, symptom scoring and thick-mount organ tissue fluorescence microscopy. Intravital TPM of the calvarial bone marrow (BM) was performed through intact bone under general anesthesia. By interline multiplexing dual femtosecond lasers with four-sensor detection for 8 distinct channels, mTurquoise2 and SHG were recorded by the same sensor at, respectively, 860 and 990 nm excitation, along with GFP, YFP, DsRed and dextran-TRITC (blood tracer). AML and T cell subsets were 3-D tracked using Imaris software. RESULTS: AML1-mTurq2 cells stably and uniformly expressed bright cyan fluorescence, suitable for intravital TPM with low incident laser powers and fast imaging rates in deep tissue locations. In C57BL6 mice, sparse AML cell clusters were found in BM perivascular spaces on day 1 after cell infusion. AML cells were slowly motile (~4 um/min) and highly proliferative, gradually filling BM spaces and emerging in other organs. T cells and CD11c dendritic cells were present in leukemic BM, and the vasculature appeared largely intact and well perfused. T cells interacted with AML cells and the stroma, migrating with high average velocities (~10 µm/min) and slowing down to ~3 µm/min in late-stage disease. After 2 days of BL-8040 treatment, disease symptom scores improved from 3 to 1 while the untreated controls progressed from 3 to 4 (range 0-6). TPM revealed a 4-fold reduction of AML cellularity in BM. Cellular velocities of both AML and T cells were unchanged by BL-8040 treatment. After acute drug administration, a fraction of stromal AML cells begun entering capillary vessel lumens by amoeboid movement. The intravasated AML cells adhered to vessel wall for 1-2 minutes before rapid detachment. Some cells remained tethered while already loose in the blood stream. CONCLUSIONS: A novel, brightly cyan-fluorescent syngeneic AML1-mTurq2 AML model is advantageous for 6-color intravital microscopy of cell trafficking and immune surveillance in optimal compatibility with green, yellow and red reporters of cell lineages and tissue architecture. Using this model, we show that CXCR4 inhibitor BL-8040 decreases AML BM cellularity by increasing the frequency of intravasation without increasing AML migratory velocity. Disclosures Zal: Daiichi-Sankyo: Research Funding; NIH-CTEP: Research Funding; BioLineRx: Research Funding; VueBio.com: Equity Ownership; NIH/NCI: Research Funding; CPRIT: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy.

Ameboid movement of cancer cells

Cell migration is a very complicated process essential for proper functioning of all living cells and organisms. It underlies numerous physiological processes as embryogenesis or wound healing as well as pathological processes such as cancer cell metastasis. The manner of cell locomotion was classified based on many parameters. There are two ways of individual migration: amoeboid and mesenchymal. The locomotion of groups of cells is known as collective type of movement. Amoeboid migration refers to rounded or ellipsoid cells and is regulated by Rho family proteins. It is stimulated by GTPase Rho and kinase ROCK. Cells which migrate in amoeboid mode do not form mature focal adhesions or stress fibres composed of polymerized actin. These cells form very dynamic migratory protrusions called blebbs. They are formed on the leading edge of the cell, which moves forward due to contractions occurring at opposite edge. In contrast to mesenchymal mode of movement, in amoeboid migration proteases activity is not required, because cells just squeeze through gaps present in extracellular matrix using actomyosin contractility. Additionally cells are able to change their mode of migration. One of this possible transformation is mesenchymal to amoeboid transition, which is crucial in metastasis and cancer invasion. This paper describes mechanisms responsible for amoeboid movement and basic pathways regulating this process.

Inseparable tandem: evolution chooses ATP and Ca 2+ to control life, death and cellular signalling

From the very dawn of biological evolution, ATP was selected as a multipurpose energy-storing molecule. Metabolism of ATP required intracellular free Ca 2+ to be set at exceedingly low concentrations, which in turn provided the background for the role of Ca 2+ as a universal signalling molecule. The early-eukaryote life forms also evolved functional compartmentalization and vesicle trafficking, which used Ca 2+ as a universal signalling ion; similarly, Ca 2+ is needed for regulation of ciliary and flagellar beat, amoeboid movement, intracellular transport, as well as of numerous metabolic processes. Thus, during evolution, exploitation of atmospheric oxygen and increasingly efficient ATP production via oxidative phosphorylation by bacterial endosymbionts were a first step for the emergence of complex eukaryotic cells. Simultaneously, Ca 2+ started to be exploited for short-range signalling, despite restrictions by the preset phosphate-based energy metabolism, when both phosphates and Ca 2+ interfere with each other because of the low solubility of calcium phosphates. The need to keep cytosolic Ca 2+ low forced cells to restrict Ca 2+ signals in space and time and to develop energetically favourable Ca 2+ signalling and Ca 2+ microdomains. These steps in tandem dominated further evolution. The ATP molecule (often released by Ca 2+ -regulated exocytosis) rapidly grew to be the universal chemical messenger for intercellular communication; ATP effects are mediated by an extended family of purinoceptors often linked to Ca 2+ signalling. Similar to atmospheric oxygen, Ca 2+ must have been reverted from a deleterious agent to a most useful (intra- and extracellular) signalling molecule. Invention of intracellular trafficking further increased the role for Ca 2+ homeostasis that became critical for regulation of cell survival and cell death. Several mutually interdependent effects of Ca 2+ and ATP have been exploited in evolution, thus turning an originally unholy alliance into a fascinating success story. This article is part of the themed issue ‘Evolution brings Ca 2+ and ATP together to control life and death’.