STING activator 2'3'-cGAMP enhanced HSV-1-based oncolytic viral therapy.

Oncolytic viruses (OVs) can selectively replicate in tumor cells and remodel the microenvironment of immunologically cold tumors, making them a promising strategy to evoke antitumor immunity. Similarly, agonists of the stimulator of interferon genes (STING)-interferon (IFN) pathway, the main cellular antiviral system, provide antitumor benefits by inducing the activation of dendritic cells (DC). Considering how the activation of the STING-IFN pathway could potentially inhibit OV replication, the use of STING agonists alongside OV therapy remains largely unexplored. Here, we explored the antitumor efficacy of combining an HSV-1-based OV, C-REV, with a membrane-impermeable STING agonist, 2'3'-GAMP. Our results demonstrated that tumor cells harbor a largely defective STING-IFN pathway, thereby preventing significant antiviral IFN induction regardless of the permeability of the STING agonist. In vivo, the combination therapy induced more proliferative KLRG1-high PD1-low CD8+ T-cells and activated CD103+ DC in the tumor site and increased tumor-specific CD44+ CD8+ T-cells in the lymph node. Overall, the combination therapy of C-REV with 2'3'-cGAMP elicited antitumor immune memory responses and significantly enhanced systemic antitumor immunity in both treated and non-treated distal tumors.

The Impact of Metformin on Tumor-Infiltrated Immune Cells: Preclinical and Clinical Studies.

The tumor microenvironment (TME) plays a pivotal role in the fate of cancer cells, and tumor-infiltrating immune cells have emerged as key players in shaping this complex milieu. Cancer is one of the leading causes of death in the world. The most common standard treatments for cancer are surgery, radiation therapy, and chemotherapeutic drugs. In the last decade, immunotherapy has had a potential effect on the treatment of cancer patients with poor prognoses. One of the immune therapeutic targeted approaches that shows anticancer efficacy is a type 2 diabetes medication, metformin. Beyond its glycemic control properties, studies have revealed intriguing immunomodulatory properties of metformin. Meanwhile, several studies focus on the impact of metformin on tumor-infiltrating immune cells in various tumor models. In several tumor models, metformin can modulate tumor-infiltrated effector immune cells, CD8+, CD4+ T cells, and natural killer (NK) cells, as well as suppressor immune cells, T regulatory cells, tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs). In this review, we discuss the role of metformin in modulating tumor-infiltrating immune cells in different preclinical models and clinical trials. Both preclinical and clinical studies suggest that metformin holds promise as adjunctive therapy in cancer treatment by modulating the immune response within the tumor microenvironment. Nonetheless, both the tumor type and the combined therapy have an impact on the specific targets of metformin in the TME. Further investigations are warranted to elucidate the precise mechanisms underlying the immunomodulatory effects of metformin and to optimize its clinical application in cancer patients.

Metformin enhances the antitumor activity of oncolytic herpes simplex virus HF10 (canerpaturev) in a pancreatic cell cancer subcutaneous model.

Oncolytic virus (OV) therapy is a promising cancer immunotherapy, especially for cold tumors by inducing the direct lysis of cancer cells and initiation of potent antitumor response. Canerpaturev (C-REV) is an attenuated oncolytic herpes simplex virus-1, which demonstrated a potent antitumor effect in various preclinical models when used either alone or combined. Metformin is a commonly prescribed antidiabetic drug that demonstrated a potent immune modulator effect and antitumor response. We combined C-REV with metformin in a low immunogenic bilateral murine tumor model to enhance C-REV's antitumor efficacy. In vitro, metformin does not enhance the C-REV cell cytotoxic effect. However, in in vivo model, intratumoral administration of C-REV with the systemic administration of metformin led to synergistic antitumor effect on both sides of tumor and prolonged survival. Moreover, combination therapy increased the effector CD44+ CD8+ PD1- subset and decreased the proportion of terminally-differentiated CD103+ KLRG-1+ T-regulatory cells on both sides of tumor. Interestingly, combination therapy efficiently modulates conventional dendritic cells type-1 (cDC1) on tumors, and tumor-drained lymph nodes. Our findings suggest that combination of C-REV and metformin enhances systemic antitumor immunity. This study may provide insights into the mechanism of action of OV therapy plus metformin combination against various tumor models.

S-1 facilitates canerpaturev (C-REV)-induced antitumor efficacy in a triple-negative breast cancer model.

Canerpaturev (C-REV) is a highly attenuated, replication-competent, mutant strain of oncolytic herpes simplex virus type 1 that may be an effective new cancer treatment option. S-1, an oral formulation containing the 5-fluorouracil (5-FU) prodrug tegafur and the two enzyme modulators gimeracil and oteracil, is used as a key chemotherapeutic agent for metastatic recurrent breast cancer. Although the antitumor effects of oncolytic viruses combined with 5-FU in vivo have been reported, the detailed mechanisms are unknown. Here, we investigated the antitumor mechanism of the combination of C-REV and S-1 in triple-negative breast cancer (TNBC) in the context of tumor immunity. The combined effect of C-REV and S-1 was evaluated in a bilateral tumor model of murine TNBC 4T1 in vivo. S-1 enhanced the TNBC growth inhibitory effects of C-REV, and decreased the number of tumor-infiltrating, myeloid-derived suppressor cells (MDSCs), which suppress both innate and adaptive immune responses. Moreover, C-REV alone and in combination with S-1 significantly increased the number of CD8+ T cells in the tumor and the production of interferon γ (IFNγ) from these cells. Our findings indicate that C-REV suppresses TNBC tumor growth by inducing the expansion of effector CD8+ T cell subsets in tumors in which S-1 can inhibit MDSC function. Our study suggests that MDSCs may be an important cellular target for breast cancer treatment. The combination of C-REV and S-1 is a new approach that might be directly translated into future clinical trials against TNBC.

C-REV Retains High Infectivity Regardless of the Expression Levels of cGAS and STING in Cultured Pancreatic Cancer Cells.

Oncolytic virus (OV) therapy is widely considered as a major breakthrough in anti-cancer treatments. In our previous study, the efficacy and safety of using C-REV for anti-cancer therapy in patients during stage I clinical trial was reported. The stimulator of interferon genes (STING)-TBK1-IRF3-IFN pathway is known to act as the central cellular host defense against viral infection. Recent reports have linked low expression levels of cGAS and STING in cancer cells to poor prognosis among patients. Moreover, downregulation of cGAS and STING has been linked to higher susceptibility to OV infection among several cancer cell lines. In this paper, we show that there is little correlation between levels of cGAS/STING expression and susceptibility to C-REV among human pancreatic cancer cell lines. Despite having a responsive STING pathway, BxPC-3 cells are highly susceptible to C-REV infection. Upon pre-activation of the STING pathway, BxPc-3 cells exhibited resistance to C-REV infection. However, without pre-activation, C-REV completely suppressed the STING pathway in BxPC-3 cells. Additionally, despite harboring defects in the STING pathway, other high-grade cancer cell lines, such as Capan-2, PANC-1 and MiaPaCa-2, still exhibited low susceptibility to C-REV infection. Furthermore, overexpression of STING in MiaPaCa-2 cells altered susceptibility to a limited extent. Taken together, our data suggest that the cGAS-STING pathway plays a minor role in the susceptibility of pancreatic cancer cell lines to C-REV infection.

Oncolytic herpes simplex virus HF10 (canerpaturev) promotes accumulation of CD8+ PD-1- tumor-infiltrating T cells in PD-L1-enriched tumor microenvironment.

Oncolytic viruses (OVs) remodel the tumor microenvironment by switching a "cold" tumor into a "hot" tumor with high CD8+ T-cell infiltration. CD8+ T-cell activity plays an essential role in the antitumor efficacy of OVs. However, the activity of T cells is impaired by the programmed cell death protein-1/programmed cell death-ligand 1 (PD-1/PD-L1) interaction. To date, it remains unclear why OVs alone have a significant antitumor activity even when PD-L1 expression persists on tumor or immune cells. In this study, we found that canerpaturev (C-REV) treatment significantly suppressed tumor growth, even though it induced a significant increase in PD-L1 expression in tumors in vivo as well as persistence of high PD-L1 expression on antigen-presenting cells (macrophage and dendritic cells [DCs]). Surprisingly, we observed that C-REV treatment increased the abundance of activated CD8+ PD-1- tumor-infiltrating lymphocytes (TILs) in the tumor on both the injected and contralateral sides, although infiltration of CD8+ PD-1high TILs into the tumor was observed in the control group. Moreover, the difference in PD-1 expression was observed only in tumors after treatment with C-REV, whereas most CD8+ T cells in the spleen, tumor-draining lymph nodes and blood were PD-1-negative, and this did not change after C-REV treatment. In addition, changes in expression of T-cell immunoglobulin and mucin-domain containing-3 and T-cell immune-receptor with Ig and ITIM domains were not observed on CD8+ TILs after C-REV treatment. Taken together, our findings may reveal mechanisms that allow OVs to trigger an antitumor immune response, irrespective of a PD-L1-enriched tumor microenvironment, by recruitment of CD8+ PD-1- TILs.

Combination of Cetuximab and Oncolytic Virus Canerpaturev Synergistically Inhibits Human Colorectal Cancer Growth.

The naturally occurring oncolytic herpes simplex virus canerpaturev (C-REV), formerly HF10, proved its therapeutic efficacy and safety in multiple clinical trials against melanoma, pancreatic, breast, and head and neck cancers. Meanwhile, patients with colorectal cancer, which has increased in prevalence in recent decades, continue to have poor prognosis and morbidity. Combination therapy has better response rates than monotherapy. Hence, we investigated the antitumor efficacy of cetuximab, a widely used anti-epidermal growth factor receptor (EGFR) monoclonal antibody, and C-REV, either alone or in combination, in vitro and in an in vivo human colorectal xenograft model. In human colorectal cancer cell lines with different levels of EGFR expression (HT-29, WiDr, and CW2), C-REV exhibited cytotoxic effects in a time- and dose-dependent manner, irrespective of EGFR expression. Moreover, cetuximab had no effect on viral replication in vitro. Combining cetuximab and C-REV induced a synergistic antitumor effect in HT-29 tumor xenograft models by promoting the distribution of C-REV throughout the tumor and suppressing angiogenesis. Application of cetuximab prior to C-REV yielded better tumor regression than administration of the drug after the virus. Thus, cetuximab represents an ideal virus-associated agent for antitumor therapy, and combination therapy represents a promising antitumor strategy for human colorectal cancer.

The Current Status and Future Prospects of Oncolytic Viruses in Clinical Trials against Melanoma, Glioma, Pancreatic, and Breast Cancers.

Oncolytic viral therapy has been accepted as a standard immunotherapy since talimogene laherparepvec (T-VEC, Imlygic®) was approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for melanoma treatment in 2015. Various oncolytic viruses (OVs), such as HF10 (Canerpaturev-C-REV) and CVA21 (CAVATAK), are now actively being developed in phase II as monotherapies, or in combination with immune checkpoint inhibitors against melanoma. Moreover, in glioma, several OVs have clearly demonstrated both safety and a promising efficacy in the phase I clinical trials. Additionally, the safety of several OVs, such as pelareorep (Reolysin®), proved their safety and efficacy in combination with paclitaxel in breast cancer patients, but the outcomes of OVs as monotherapy against breast cancer have not provided a clear therapeutic strategy for OVs. The clinical trials of OVs against pancreatic cancer have not yet demonstrated efficacy as either monotherapy or as part of combination therapy. However, there are several oncolytic viruses that have successfully proved their efficacy in different preclinical models. In this review, we mainly focused on the oncolytic viruses that transitioned into clinical trials against melanoma, glioma, pancreatic, and breast cancers. Hence, we described the current status and future prospects of OVs clinical trials against melanoma, glioma, pancreatic, and breast cancers.

Publisher Correction: Early lineage segregation of multipotent embryonic mammary gland progenitors.

In the version of this Article originally published, ref. 52 was incorrectly only attributed to its corresponding author, Fre, S., and an older title was used. The correct citation should have been: Lilja, A. M. et al. Clonal analysis of Notch1-expressing cells reveals the existence of unipotent stem cells that retain long-term plasticity in the embryonic mammary gland. Nat. Cell Biol. https://doi.org/10.1038/s41556-018-0108-1 (2018)'. This has now been amended in all online versions of the Article.

Early lineage segregation of multipotent embryonic mammary gland progenitors.

The mammary gland is composed of basal cells and luminal cells. It is generally believed that the mammary gland arises from embryonic multipotent progenitors, but it remains unclear when lineage restriction occurs and what mechanisms are responsible for the switch from multipotency to unipotency during its morphogenesis. Here, we perform multicolour lineage tracing and assess the fate of single progenitors, and demonstrate the existence of a developmental switch from multipotency to unipotency during embryonic mammary gland development. Molecular profiling and single cell RNA-seq revealed that embryonic multipotent progenitors express a unique hybrid basal and luminal signature and the factors associated with the different lineages. Sustained p63 expression in embryonic multipotent progenitors promotes unipotent basal cell fate and was sufficient to reprogram adult luminal cells into basal cells by promoting an intermediate hybrid multipotent-like state. Altogether, this study identifies the timing and the mechanisms mediating early lineage segregation of multipotent progenitors during mammary gland development.

Tbx3-dependent amplifying stem cell progeny drives interfollicular epidermal expansion during pregnancy and regeneration.

The skin surface area varies flexibly in response to body shape changes. Skin homeostasis is maintained by stem cells residing in the basal layer of the interfollicular epidermis. However, how the interfollicular epidermal stem cells response to physiological body shape changes remains elusive. Here, we identify a highly proliferative interfollicular epidermal basal cell population in the rapidly expanding abdominal skin of pregnant mice. These cells express Tbx3 that is necessary for their propagation to drive skin expansion. The Tbx3+ basal cells are generated from Axin2+ interfollicular epidermal stem cells through planar-oriented asymmetric or symmetric cell divisions, and express transit-amplifying cell marker CD71. This biased division of Axin2+ interfollicular epidermal stem cells is induced by Sfrp1 and Igfbp2 proteins secreted from dermal cells. The Tbx3+ basal cells promote wound repair, which is enhanced by Sfrp1 and Igfbp2. This study elucidates the interfollicular epidermal stem cell/progeny organisation during pregnancy and suggests its application in regenerative medicine.The abdominal skin expands rapidly during pregnancy. Here the authors show that a population of highly proliferative stem cell progenies expressing the transcription factor Tbx3 is required for abdominal skin expansion in pregnant mice.

Katanin p80, NuMA and cytoplasmic dynein cooperate to control microtubule dynamics.

Human mutations in KATNB1 (p80) cause severe congenital cortical malformations, which encompass the clinical features of both microcephaly and lissencephaly. Although p80 plays critical roles during brain development, the underlying mechanisms remain predominately unknown. Here, we demonstrate that p80 regulates microtubule (MT) remodeling in combination with NuMA (nuclear mitotic apparatus protein) and cytoplasmic dynein. We show that p80 shuttles between the nucleus and spindle pole in synchrony with the cell cycle. Interestingly, this striking feature is shared with NuMA. Importantly, p80 is essential for aster formation and maintenance in vitro. siRNA-mediated depletion of p80 and/or NuMA induced abnormal mitotic phenotypes in cultured mouse embryonic fibroblasts and aberrant neurogenesis and neuronal migration in the mouse embryonic brain. Importantly, these results were confirmed in p80-mutant harboring patient-derived induced pluripotent stem cells and brain organoids. Taken together, our findings provide valuable insights into the pathogenesis of severe microlissencephaly, in which p80 and NuMA delineate a common pathway for neurogenesis and neuronal migration via MT organization at the centrosome/spindle pole.

Interphase adhesion geometry is transmitted to an internal regulator for spindle orientation via caveolin-1.

Despite theoretical and physical studies implying that cell-extracellular matrix adhesion geometry governs the orientation of the cell division axis, the molecular mechanisms that translate interphase adhesion geometry to the mitotic spindle orientation remain elusive. Here, we show that the cellular edge retraction during mitotic cell rounding correlates with the spindle axis. At the onset of mitotic cell rounding, caveolin-1 is targeted to the retracting cortical region at the proximal end of retraction fibres, where ganglioside GM1-enriched membrane domains with clusters of caveola-like structures are formed in an integrin and RhoA-dependent manner. Furthermore, Gαi1-LGN-NuMA, a well-known regulatory complex of spindle orientation, is targeted to the caveolin-1-enriched cortical region to guide the spindle axis towards the cellular edge retraction. We propose that retraction-induced cortical heterogeneity of caveolin-1 during mitotic cell rounding sets the spindle orientation in the context of adhesion geometry.

PCTK1 regulates integrin-dependent spindle orientation via protein kinase A regulatory subunit KAP0 and myosin X.

Integrin-dependent cell-extracellular matrix (ECM) adhesion is a determinant of spindle orientation. However, the signaling pathways that couple integrins to spindle orientation remain elusive. Here, we show that PCTAIRE-1 kinase (PCTK1), a member of the cyclin-dependent kinases (CDKs) whose function is poorly characterized, plays an essential role in this process. PCTK1 regulates spindle orientation in a kinase-dependent manner. Phosphoproteomic analysis together with an RNA interference screen revealed that PCTK1 regulates spindle orientation through phosphorylation of Ser83 on KAP0, a regulatory subunit of protein kinase A (PKA). This phosphorylation is dispensable for KAP0 dimerization and for PKA binding but is necessary for its interaction with myosin X, a regulator of spindle orientation. KAP0 binds to the FERM domain of myosin X and enhances the association of myosin X-FERM with ß1 integrin. This interaction between myosin X-FERM and ß1 integrin appeared to be crucial for spindle orientation control. We propose that PCTK1-KAP0-myosin X-ß1 integrin is a functional module providing a link between ECM and the actin cytoskeleton in the ECM-dependent control of spindle orientation.

Pregnenolone functions in centriole cohesion during mitosis.

Cell division is controlled by a multitude of protein enzymes, but little is known about roles of metabolites in this mechanism. Here, we show that pregnenolone (P5), a steroid that is produced from cholesterol by the steroidogenic enzyme Cyp11a1, has an essential role in centriole cohesion during mitosis. During prometa-metaphase, P5 is accumulated around the spindle poles. Depletion of P5 induces multipolar spindles that result from premature centriole disengagement, which are rescued by ectopic introduction of P5, but not its downstream metabolites, into the cells. Premature centriole disengagement, induced by loss of P5, is not a result of precocious activation of separase, a key factor for the centriole disengagement in anaphase. Rather, P5 directly binds to the N-terminal coiled-coil domain of short-form of shugoshin 1 (sSgo1), a protector for centriole cohesion and recruits it to spindle poles in mitosis. Our results thus reveal a steroid-mediated centriole protection mechanism.

Inhibition of endocytic vesicle fusion by Plk1-mediated phosphorylation of vimentin during mitosis.

Endocytic vesicle fusion is inhibited during mitosis, but the molecular pathways that mediate the inhibition remain unclear. Here we uncovered an essential role of Polo-like kinase 1 (Plk1) in this mechanism. Phosphoproteomic analysis revealed that Plk1 phosphorylates the intermediate filament protein vimentin on Ser459, which is dispensable for its filament formation but is necessary for the inhibition of endocytic vesicle fusion in mitosis. Furthermore, this mechanism is required for integrin trafficking toward the cleavage furrow during cytokinesis. Our results thus identify a novel mechanism for fusion inhibition in mitosis and implicate its role in vesicle trafficking after anaphase onset.

ABL1 joins the cadre of spindle orientation machinery.

Directing the axis of cell division toward extrinsic and intrinsic cues plays a fundamental role in morphogenesis, asymmetric cell division, and stem cell self-renewal. Recent studies highlight the misorientation of the cell division axis as a cause of mammalian diseases, including polycystic kidney disease. Although the core regulators for oriented cell division have been identified in invertebrate model systems, we still have an imprecise picture of the relevant signaling networks in the mammalian system. The reasons for this include the lack of established approaches in mammalian cells to survey the molecules required for the spindle orientation. Here we summarize our recent study on a genome-scale RNA-mediated interference screen of human kinases to identify a new player for the oriented cell division in both culture cells and developing mammalian tissues.

ABL1 regulates spindle orientation in adherent cells and mammalian skin.

Despite the growing evidence for the regulated spindle orientation in mammals, a systematic approach for identifying the responsible genes in mammalian cells has not been established. Here we perform a kinase-targeting RNAi screen in HeLa cells and identify ABL1 as a novel regulator of spindle orientation. Knockdown of ABL1 causes the cortical accumulation of Leu-Gly-Asn repeat-enriched-protein (LGN), an evolutionarily conserved regulator of spindle orientation. This results in the LGN-dependent spindle rotation and spindle misorientation. In vivo inactivation of ABL1 by a pharmacological inhibitor or by ablation of the abl1 gene causes spindle misorientation and LGN mislocalization in mouse epidermis. Furthermore, ABL1 directly phosphorylates NuMA, a binding partner of LGN, on tyrosine 1774. This phosphorylation maintains the cortical localization of NuMA during metaphase, and ensures the LGN/NuMA-dependent spindle orientation control. This study provides a novel approach to identify genes regulating spindle orientation in mammals and uncovers new signalling pathways for this mechanism.

Revolving movement of a dynamic cluster of actin filaments during mitosis.

The actin cytoskeleton undergoes rapid changes in its architecture during mitosis. Here, we demonstrate novel actin assembly dynamics in M phase. An amorphous cluster of actin filaments appears during prometaphase, revolves horizontally along the cell cortex at a constant angular speed, and fuses into the contractile ring after three to four revolutions. Cdk1 activity is required for the formation of this mitotic actin cluster and its revolving movement. Rapid turnover of actin in the filaments takes place everywhere in the cluster and is also required for its cluster rotation during mitosis. Knockdown of Arp3, a component of the actin filament-nucleating Arp2/3 complex, inhibits the formation of the mitotic actin cluster without affecting other actin structures. These results identify Arp2/3 complex as a key factor in the generation of the dynamic actin cluster during mitosis.

PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells.

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.