Cdc42GAP deficiency contributes to the Alzheimer's disease phenotype.

Alzheimer's disease, the most common cause of dementia, is a chronic degenerative disease with typical pathological features of extracellular senile plaques and intracellular neurofibrillary tangles and a significant decrease in the density of neuronal dendritic spines. Cdc42 is a member of the small G protein family that plays an important role in regulating synaptic plasticity and is regulated by Cdc42GAP, which switches Cdc42 from active GTP-bound to inactive GDP-bound states regulating downstream pathways via effector proteins. However, few studies have focused on Cdc42 in the progression of Alzheimer's disease. In a heterozygous Cdc42GAP mouse model that exhibited elevated Cdc42-GTPase activity accompanied by increased Cdc42-PAK1-cofilin signalling, we found impairments in cognitive behaviours, neuron senescence, synaptic loss with depolymerization of F-actin and the pathological phenotypes of Alzheimer's disease, including phosphorylated tau (p-T231, AT8), along with increased soluble and insoluble Aß1-42 and Aß1-40, which are consistent with typical Alzheimer's disease mice. Interestingly, these impairments increased significantly with age. Furthermore, the results of quantitative phosphoproteomic analysis of the hippocampus of 11-month-old GAP mice suggested that Cdc42GAP deficiency induces and accelerates Alzheimer's disease-like phenotypes through activation of GSK-3ß by dephosphorylation at Ser9, Ser389 and/or phosphorylation at Tyr216. In addition, overexpression of dominant-negative Cdc42 in the primary hippocampal and cortical neurons of heterozygous Cdc42GAP mice reversed synaptic loss and tau hyperphosphorylation. Importantly, the Cdc42 signalling pathway, Aß1-42, Aß1-40 and GSK-3ß activity were increased in the cortical sections of Alzheimer's disease patients compared with those in healthy controls. Together, these data indicated that Cdc42GAP is involved in regulating Alzheimer's disease-like phenotypes such as cognitive deficits, dendritic spine loss, phosphorylated tau (p-T231, AT8) and increased soluble and insoluble Aß1-42 and Aß1-40, possibly through the activation of GSK-3ß, and these impairments increased significantly with age. Thus, we provide the first evidence that Cdc42 is involved in the progression of Alzheimer's disease-like phenotypes, which may provide new targets for Alzheimer's disease treatment.

Adaptive responses to mTOR gene targeting in hematopoietic stem cells reveal a proliferative mechanism evasive to mTOR inhibition.

The mechanistic target of rapamycin (mTOR) is a central regulator of cell growth and an attractive anticancer target that integrates diverse signals to control cell proliferation. Previous studies using mTOR inhibitors have shown that mTOR targeting suppresses gene expression and cell proliferation. To date, however, mTOR-targeted therapies in cancer have seen limited efficacy, and one key issue is related to the development of evasive resistance. In this manuscript, through the use of a gene targeting mouse model, we have found that inducible deletion of mTOR in hematopoietic stem cells (HSCs) results in a loss of quiescence and increased proliferation. Adaptive to the mTOR loss, mTOR-/- HSCs increase chromatin accessibility and activate global gene expression, contrary to the effects of short-term inhibition by mTOR inhibitors. Mechanistically, such genomic changes are due to a rewiring and adaptive activation of the ERK/MNK/eIF4E signaling pathway that enhances the protein translation of RNA polymerase II, which in turn leads to increased c-Myc gene expression, allowing the HSCs to thrive despite the loss of a functional mTOR pathway. This adaptive mechanism can also be utilized by leukemia cells undergoing long-term mTOR inhibitor treatment to confer resistance to mTOR drug targeting. The resistance can be counteracted by MNK, CDK9, or c-Myc inhibition. These results provide insights into the physiological role of mTOR in mammalian stem cell regulation and implicate a mechanism of evasive resistance in the context of mTOR targeting.

mDia1 and Cdc42 Regulate Activin B-Induced Migration of Bone Marrow-Derived Mesenchymal Stromal Cells.

In a previous study, we have shown that Activin B is a potent chemoattractant for bone marrow-derived mesenchymal stromal cells (BMSCs). As such, the combination of Activin B and BMSCs significantly accelerated rat skin wound healing. In another study, we showed that RhoA activation plays a key role in Activin B-induced BMSC migration. However, the role of the immediate downstream effectors of RhoA in this process is unclear. Here, we demonstrated that mammalian homolog of Drosophila diaphanous-1 (mDia1), a downstream effector of RhoA, exerts a crucial function in Activin B-induced BMSC migration by promoting membrane ruffling, microtubule morphology, and adhesion signaling dynamics. Furthermore, we showed that Activin B does not change Rac1 activity but increases Cdc42 activity in BMSCs. Inactivation of Cdc42 inhibited Activin B-stimulated Golgi reorientation and the cell migration of BMSCs. Furthermore, knockdown of mDia1 affected Activin B-induced BMSC-mediated wound healing in vivo. In conclusion, this study demonstrated that the RhoA-mDia1 and Cdc42 pathways regulate Activin B-induced BMSC migration. This study may help to optimize clinical MSC-based transplantation strategies to promote skin wound healing. Stem Cells 2019;37:150-162.

Reciprocal Regulation of Glycolysis-Driven Th17 Pathogenicity and Regulatory T Cell Stability by Cdc42.

A balance between Th17 cells and regulatory T cells (Tregs) is important for host immunity and immune tolerance. The underlying molecular mechanisms remain poorly understood. Here we have identified Cdc42 as a central regulator of Th17/Treg balance. Deletion of Cdc42 in T cells enhanced Th17 differentiation but diminished induced Treg differentiation and suppressive function. Treg-specific deletion of Cdc42 decreased natural Tregs but increased effector T cells including Th17 cells. Notably, Cdc42-deficient Th17 cells became pathogenic associated with enhanced glycolysis and Cdc42-deficient Tregs became unstable associated with weakened glycolytic signaling. Inhibition of glycolysis in Cdc42-deficient Th17 cells diminished their pathogenicity and restoration of glycolysis in Cdc42-deficient Tregs rescued their instability. Intriguingly, Cdc42 deficiency in T cells led to exacerbated wasting disease in mouse models of colitis and Treg-specific deletion of Cdc42 caused early, fatal lymphoproliferative diseases. In summary, we show that Cdc42 is a bona fide regulator of peripheral tolerance through suppression of Th17 aberrant differentiation/pathogenicity and promotion of Treg differentiation/stability/function involving metabolic signaling and thus Cdc42 pathway might be harnessed in autoimmune disease therapy.

Rational targeting Cdc42 restrains Th2 cell differentiation and prevents allergic airway inflammation.

BACKGROUND: Asthma is an allergic airway inflammation-driven disease that affects more than 300 million people world-wide. Targeted therapies for asthma are largely lacking. Although asthma symptoms can be prevented from worsening, asthma development cannot be prevented. Cdc42 GTPase has been shown to regulate actin cytoskeleton, cell proliferation and survival. OBJECTIVES: To investigate the role and targeting of Cdc42 in Th2 cell differentiation and Th2-mediated allergic airway inflammation. METHODS: Post-thymic Cdc42-deficient mice were generated by crossing Cdc42flox/flox mice with dLckicre transgenic mice in which Cre expression is driven by distal Lck promoter. Effects of post-thymic Cdc42 deletion and pharmacological targeting Cdc42 on Th2 cell differentiation were evaluated in vitro under Th2-polarized culture conditions. Effects of post-thymic Cdc42 deletion and pharmacological targeting Cdc42 on allergic airway inflammation were evaluated in ovalbumin- and/or house dust mite-induced mouse models of asthma. RESULTS: Post-thymic deletion of Cdc42 led to reduced peripheral CD8+ T cells and attenuated Th2 cell differentiation, with no effect on closely related Th1, Th17 and induced regulatory T (iTreg) cells. Post-thymic Cdc42 deficiency ameliorated allergic airway inflammation. The selective inhibition of Th2 cell differentiation by post-thymic deletion of Cdc42 was recapitulated by pharmacological targeting of Cdc42 with CASIN, a Cdc42 activity-specific chemical inhibitor. CASIN also alleviated allergic airway inflammation. CASIN-treated Cdc42-deficient mice showed comparable allergic airway inflammation to vehicle-treated Cdc42-deficient mice, indicative of negligible off-target effect of CASIN. CASIN had no effect on established allergic airway inflammation. CONCLUSION AND CLINICAL RELEVANCE: Cdc42 is required for Th2 cell differentiation and allergic airway inflammation, and rational targeting Cdc42 may serve as a preventive but not therapeutic approach for asthma control.

PKCλ/ι regulates Th17 differentiation and house dust mite-induced allergic airway inflammation.

Asthma is a chronic airway inflammation in which Th2 and Th17 cells play critical roles in its pathogenesis. We have reported that atypical protein kinase (PKC) λ/ι is a new regulator for Th2 differentiation and function. However, the role of PKCλ/ι for Th17 cells remains elusive. In this study, we explored the effect of PKCλ/ι on Th17 cells in the context of ex vivo cell culture systems and an in vivo murine model of allergic airway inflammation with the use of activated T cell-specific conditional PKCλ/ι-deficient mice. Our findings indicate that PKCλ/ι regulates Th17 cells. The secretion of Th17 effector cytokines, including IL-17, IL-21 and IL-22, were inhibited from PKCλ/ι-deficient T cells under non-skewing or Th17-skewing culture conditions. Moreover, the impaired Th17 differentiation and function by the PKCλ/ι-deficiency was associated with the downregulation of Stat3 and Rorγt, key Th17 transcription factors. We developed a model of Th17 and neutrophil-involved allergic airway inflammation by intratracheal inoculation of house dust mites. PKCλ/ι-deficiency significantly inhibited airway inflammations. The infiltrating cells in the lungs and bronchoalveolar lavage fluids were significantly reduced in conditional PKCλ/ι-deficient mice. Th17 effector cytokines were reduced in the bronchoalveolar lavage fluids and lungs at protein and mRNA levels. Thus, PKCλ/ι emerges as a critical regulator of Th17 differentiation and allergic airway hyperresponsiveness.

Endothelial Cdc42 deficiency impairs endothelial regeneration and vascular repair after inflammatory vascular injury.

BACKGROUND: Endothelial cell (EC) regeneration is essential for inflammation resolution and vascular integrity recovery after inflammatory vascular injury. Cdc42 is a central regulator of cell survival and vessel formation in EC development. However, it is unknown that whether Cdc42 could be a regulating role of EC repair following the inflammatory injury in the lung. The study sought to test the hypothesis that Cdc42 is required for endothelial regeneration and vascular integrity recovery after LPS-induced inflammatory injury. METHODS AND RESULTS: The role of Cdc42 for the regulation of pulmonary vascular endothelial repair was tested in vitro and in vivo. In LPS-induced acute lung injury (ALI) mouse models, knockout of the Cdc42 gene in ECs increased inflammatory cell infiltration and pulmonary vascular leakage and inhibited vascular EC proliferation, which eventually resulted in more severe inflammatory lung injury. In addition, siRNA-mediated knockdown of Cdc42 protein on ECs disrupted cell proliferation and migration and tube formation, which are necessary processes for recovery after inflammatory vascular injury, resulting in inflammatory vascular injury recovery defects. CONCLUSION: We found that Cdc42 deficiency impairs EC function and regeneration, which are crucial in the post-inflammatory vascular injury repair process. These findings indicate that Cdc42 is a potential target for novel treatments designed to facilitate endothelial regeneration and vascular repair in inflammatory pulmonary vascular diseases, such as ALI/ARDS.

RhoA regulates Activin B-induced stress fiber formation and migration of bone marrow-derived mesenchymal stromal cell through distinct signaling.

BACKGROUND: In our previous study, Activin B induced actin stress fiber formation and cell migration in Bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. However, the underlying molecular mechanisms are not well studied. RhoA is recognized to play a critical role in the regulation of actomyosin cytoskeletal organization and cell migration. METHODS: Pull-down assay was performed to investigate the activity of RhoA. The dominant-negative mutants of RhoA (RhoA(N19)) was used to determine whether RhoA has a role in Activin B-induced cytoskeleton organization and cell migration in BMSCs. Cytoskeleton organization was examined by fluorescence Rhodamine-phalloidin staining, and cell migration by transwell and cell scratching assay. Western blot was carried out to investigate downstream signaling cascade of RhoA. Inhibitor and siRNAs were used to detect the role of downstream signaling in stress fiber formation and/or cell migration. RESULTS: RhoA was activated by Activin B in BMSCs. RhoA(N19) blocked Activin B-induced stress fiber formation and cell migration. ROCK inhibitor blocked Activin B-induced stress fiber formation but enhanced BMSCs migration. Activin B induced phosphorylation of LIMK2 and Cofilin, which was abolished by ROCK inhibition. Both of siRNA LIMK2 and siRNA Cofilin inhibited Activin B-induced stress fiber formation. CONCLUSIONS: RhoA regulates Activin B-induced stress fiber formation and migration of BMSCs. A RhoA-ROCK-LIMK2-Cofilin signaling node exists and regulates actin stress fiber formation. RhoA regulates Activin B-induced cell migration independent of ROCK. GENERAL SIGNIFICANCE: Better understanding of the molecular mechanisms of BMSCs migration will help optimize therapeutic strategy to target BMSCs at injured tissues.

Cocaine-induced synaptic structural modification is differentially regulated by dopamine D1 and D3 receptors-mediated signaling pathways.

Synaptic plasticity plays a critical role in cocaine addiction. The dopamine D1 and D3 receptors differentially regulate the cocaine-induced gene expression, structural remodeling and behavioral response. However, how these two receptors coordinately mediate the ultra-structural changes of synapses after cocaine exposure and whether these changes are behaviorally relevant are still not clear. Here, using quantitative electron microscopy, we show that D1 and D3 receptors have distinct roles in regulating cocaine-induced ultra-structural changes of synapses in the nucleus accumbens and caudoputamen. Pre-treatment of cocaine-treated mice with D3 receptor antagonist NGB2904 resulted in an increase in the ratio of total and asymmetric synapse to neuron and in the length of postsynaptic densities, compared with cocaine treatment alone. In contrast, pre-treatment of cocaine-treated mice with D1 receptor antagonist SCH23390 caused a reduction in synapse-to-neuron ratio and in postsynaptic densities length. Similarly, NGB2904 and SCH23390 showed opposite/differential effects on cocaine-induced structural plasticity, conditioned place preference and locomotor activity and signaling activation, including the activation of ERK, CREB and NR1 and the expression of c-fos and Cdk5. Therefore, we provide direct electron microscopy evidence that dopamine D1 and D3 receptors reciprocally regulate the ultra-structural changes of synapses following chronic exposure to cocaine. In addition, our data suggest that D1 and D3 receptors may regulate cocaine-induced ultra-structural changes and behavior responses by impact on structural plasticity and signaling transduction.

RhoA orchestrates glycolysis for TH2 cell differentiation and allergic airway inflammation.

BACKGROUND: Mitochondrial metabolism is known to be important for T-cell activation. However, its involvement in effector T-cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T-cell activation and effector cell differentiation and function remains largely unknown. OBJECTIVE: We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for TH2 cell differentiation and TH2-mediated allergic airway inflammation. METHODS: Conditional RhoA-deficient mice were generated by crossing RhoA(flox/flox) mice with CD2-Cre transgenic mice. Effects of RhoA on TH2 differentiation were evaluated based on in vitro TH2-polarized culture conditions and in vivo in ovalbumin-induced allergic airway inflammation. Cytokine levels were measured by using intracellular staining and ELISA. T-cell metabolism was measured by using the Seahorse XF24 Analyzer and flow cytometry. RESULTS: Disruption of RhoA inhibited T-cell activation and TH2 differentiation in vitro and prevented the development of allergic airway inflammation in vivo, with no effect on TH1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways, such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to TH2 cell differentiation and allergic airway inflammation through regulating IL-4 receptor mRNA expression and TH2-specific signaling events. Finally, inhibition of Rho-associated protein kinase, an immediate downstream effector of RhoA, blocked TH2 differentiation and allergic airway inflammation. CONCLUSION: RhoA is a key component of the signaling cascades leading to TH2 differentiation and allergic airway inflammation at least in part through control of T-cell metabolism and the Rho-associated protein kinase pathway.

Gene targeting RhoA reveals its essential role in coordinating mitochondrial function and thymocyte development.

Thymocyte development is regulated by complex signaling pathways. How these signaling cascades are coordinated remains elusive. RhoA of the Rho family small GTPases plays an important role in actin cytoskeleton organization, cell adhesion, migration, proliferation, and survival. Nonetheless, the physiological function of RhoA in thymocyte development is not clear. By characterizing a conditional gene targeting mouse model bearing T cell deletion of RhoA, we show that RhoA critically regulates thymocyte development by coordinating multiple developmental events. RhoA gene disruption caused a strong developmental block at the pre-TCR checkpoint and during positive selection. Ablation of RhoA led to reduced DNA synthesis in CD4(-)CD8(-), CD4(+)CD8(-), and CD4(-)CD8(+) thymocytes but not in CD4(+)CD8(+) thymocytes. Instead, RhoA-deficient CD4(+)CD8(+) thymocytes showed an impaired mitosis. Furthermore, we found that abrogation of RhoA led to an increased apoptosis in all thymocyte subpopulations. Importantly, we show that the increased apoptosis was resulted from reduced pre-TCR expression and increased production of reactive oxygen species (ROS), which may be because of an enhanced mitochondrial function, as manifested by increased oxidative phosphorylation, glycolysis, mitochondrial membrane potential, and mitochondrial biogenesis in RhoA-deficient thymocytes. Restoration of pre-TCR expression or treatment of RhoA-deficient mice with a ROS scavenger N-acetylcysteine partially restored thymocyte development. These results suggest that RhoA is required for thymocyte development and indicate, to our knowledge, for the first time that fine-tuning of ROS production by RhoA, through a delicate control of metabolic circuit, may contribute to thymopoiesis.

Cocaine activates Rac1 to control structural and behavioral plasticity in caudate putamen.

Repeated exposure to cocaine was previously found to cause sensitized behavioral responses and structural remodeling on medium spiny neurons of the nucleus accumbens (NAc) and caudate putamen (CPu). Rac1 has emerged as a key integrator of environmental cues that regulates dendritic cytoskeletons. In this study, we investigated the role of Rac1 in cocaine-induced dendritic and behavioral plasticity in the CPu. We found that Rac1 activation was reduced in the NAc but increased in the CPu following repeated cocaine treatment. Inhibition of Rac1 activity by a Rac1-specific inhibitor NSC23766, overexpression of a dominant negative mutant of Rac1 (T17N-Rac1) or local knockout of Rac1 attenuated the cocaine-induced increase in dendrites and spine density in the CPu, whereas overexpression of a constitutively active Rac1 exert the opposite effect. Moreover, NSC23766 reversed the increased number of asymmetric spine synapses in the CPu following chronic cocaine exposure. Downregulation of Rac1 activity likewise attenuates behavioral reward responses to cocaine exposure, with activation of Rac1 producing the opposite effect. Thus, Rac1 signaling is differentially regulated in the NAc and CPu after repeated cocaine treatment, and induction of Rac1 activation in the CPu is important for cocaine exposure-induced dendritic remodeling and behavioral plasticity.

Cdc42 regulates neutrophil migration via crosstalk between WASp, CD11b, and microtubules.

Chemotaxis promotes neutrophil participation in cellular defense by enabling neutrophil migration to infected tissue and is controlled by persistent cell polarization. One long-standing question of neutrophil polarity has been how the pseudopod and the uropod are coordinated. In our previous report, we suggested that Rho GTPase Cdc42 controls neutrophil polarity through CD11b signaling at the uropod, albeit through an unknown mechanism. Here, we show that Cdc42 controls polarity, unexpectedly, via its effector WASp. Cdc42 controls WASp activation and its distant localization to the uropod. At the uropod, WASp regulates the reorganization of CD11b integrin into detergent resistant membrane domains; in turn, CD11b recruits the microtubule end binding protein EB1 to capture and stabilize microtubules at the uropod. This organization is necessary to maintain neutrophil polarity during migration and is critical for neutrophil emigration into inflamed lungs. These results suggest unrecognized mechanism of neutrophil polarity in which WASp mediates long-distance control of the uropod by Cdc42 to maintain a proper balance between the pseudopod and the uropod. Our study reveals a new function for WASp in the control of neutrophil polarity via crosstalk between CD11b and microtubules.

Efficacy and advantage of immunotherapy for melanoma via intramuscular co-expression of plasmid-encoded PD-1 and CTLA-4 scFvs.

Immunotherapy, in the shape of immune checkpoint inhibitors (ICIs), has completely changed the treatment of cancer. However, the increasing expense of treatment and the frequency of immune-related side effects, which are frequently associated with combination antibody therapies and Fc fragment of antibody, have limited the patient's ability to benefit from these treatments. Herein, we presented the therapeutic effects of the plasmid-encoded PD-1 and CTLA-4 scFvs (single-chain variable fragment) for melanoma via an optimized intramuscular gene delivery system. After a single injection, the plasmid-encoded ICI scFv in mouse sera continued to be above 150 ng/mL for 3 weeks and reached peak amounts of 600 ng/mL. Intramuscular delivery of plasmid encoding PD-1 and CTLA-4 scFvs significantly changed the tumor microenvironment, delayed tumor growth, and prolonged survival in melanoma-bearing mice. Furthermore, no significant toxicity was observed, suggesting that this approach could improve the biosafety of ICIs combination therapy. Overall, the expression of ICI scFvs in vivo using intramuscular plasmid delivery could potentially develop into a reliable, affordable, and safe immunotherapy technique, expanding the range of antibody-based gene therapy systems that are available.

Mouse gene targeting reveals an essential role of mTOR in hematopoietic stem cell engraftment and hematopoiesis.

mTOR integrates signals from nutrients and growth factors to control protein synthesis, cell growth, and survival. Although mTOR has been established as a therapeutic target in hematologic malignancies, its physiological role in regulating hematopoiesis remains unclear. Here we show that conditional gene targeting of mTOR causes bone marrow failure and defects in multi-lineage hematopoiesis including myelopoiesis, erythropoiesis, thrombopoiesis, and lymphopoiesis. mTOR deficiency results in loss of quiescence of hematopoietic stem cells, leading to a transient increase but long-term exhaustion and defective engraftment of hematopoietic stem cells in lethally irradiated recipient mice. Furthermore, ablation of mTOR causes increased apoptosis in lineage-committed blood cells but not hematopoietic stem cells, indicating a differentiation stage-specific function. These results demonstrate that mTOR is essential for hematopoietic stem cell engraftment and multi-lineage hematopoiesis.

Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis.

T-cell homeostasis is essential for normal functioning of the immune system. IL-7 receptor (IL-7R) and T-cell receptor (TCR) signaling are pivotal for T-cell homeostatic regulation. The detailed mechanisms regulating T-cell homeostasis and how IL-7R and TCR signaling are coordinated are largely unknown. Here we demonstrate that T cell-specific deletion of cell-division cycle 42 (Cdc42) GTPase causes a profound loss of mature T cells. Deletion of Cdc42 leads to a markedly increased expression of growth factor independence-1 (Gfi-1) and represses expression of IL-7Rα. In the absence of Cdc42, aberrant ERK1/2 MAP kinase activity results in enhanced, TCR-mediated T-cell proliferation. In vivo reconstitution of effector-binding-defective Cdc42 mutants and the effector p21 protein-activated kinase 1 (PAK1) into Cdc42-deficient T cells showed that PAK1 is both necessary and sufficient for Cdc42-regulated T-cell homeostasis. Thus, T-cell homeostasis is maintained through a concerted regulation of Gfi-1-IL-7R-controlled cytokine responsiveness and ERK-mediated TCR signaling strength by the Cdc42-PAK1 signaling axis.

Mechanism of inert inflammation in an immune checkpoint blockade-resistant tumor subtype bearing transcription elongation defects.

The clinical response to immune checkpoint blockade (ICB) correlates with tumor-infiltrating cytolytic T lymphocytes (CTLs) prior to treatment. However, many of these inflamed tumors resist ICB through unknown mechanisms. We show that tumors with transcription elongation deficiencies (TEdef+), which we previously reported as being resistant to ICB in mouse models and the clinic, have high baseline CTLs. We show that high baseline CTLs in TEdef+ tumors result from aberrant activation of the nucleic acid sensing-TBK1-CCL5/CXCL9 signaling cascade, which results in an immunosuppressive microenvironment with elevated regulatory T cells and exhausted CTLs. ICB therapy of TEdef+ tumors fail to increase CTL infiltration and suppress tumor growth in both experimental and clinical settings, suggesting that TEdef+, along with surrogate markers of tumor immunogenicity such as tumor mutational burden and CTLs, should be considered in the decision process for patient immunotherapy indication.

Kinase-independent role of mTOR and on-/off-target effects of an mTOR kinase inhibitor.

mTOR, as a serine/threonine kinase, is a widely pursued anticancer target. Multiple clinical trials of mTOR kinase inhibitors are ongoing, but their specificity and safety features remain lacking. Here, we have employed an inducible kinase-inactive D2338A mTOR knock-in mouse model (mTOR-/KI) together with a mTOR conditional knockout model (mTOR-/-) to assess the kinase-dependent/-independent function of mTOR in hematopoiesis and the on-/off-target effects of mTOR kinase inhibitor AZD2014. Despite exhibiting many similar phenotypes to mTOR-/- mice in hematopoiesis, the mTOR-/KI mice survived longer and showed differences in hematopoietic progenitor cells compared to mTOR-/- mice, suggesting a kinase-independent function of mTOR in hematopoiesis. Gene expression signatures in hematopoietic stem cells (HSCs) further revealed both kinase-dependent and independent effects of mTOR. AZD2014, a lead mTOR kinase inhibitor, appeared to work mostly on-target in suppressing mTOR kinase activity, mimicking that of mTOR-/KI HSCs in transcriptome analysis, but it also induced a small set of off-target responses in mTOR-/KI HSCs. In murine and human myeloid leukemia, besides kinase-inhibitory on-target effects, AZD2014 displayed similar off-target and growth-inhibitory cytostatic effects. These studies provide new insights into kinase-dependent/-independent effects of mTOR in hematopoiesis and present a genetic means for precisely assessing the specificity of mTOR kinase inhibitors.

Cdc42 signaling regulated by dopamine D2 receptor correlatively links specific brain regions of hippocampus to cocaine addiction.

BACKGROUND: Hippocampus plays critical roles in drug addiction. Cocaine-induced modifications in dopamine receptor function and the downstream signaling are important regulation mechanisms in cocaine addiction. Rac regulates actin filament accumulation while Cdc42 stimulates the formation of filopodia and neurite outgrowth. Based on the region specific roles of small GTPases in brain, we focused on the hippocampal subregions to detect the regulation of Cdc42 signaling in long-term morphological and behavioral adaptations to cocaine. METHODS: Genetically modified mouse models of Cdc42, dopamine receptor D1 (D1R) and D2 (D2R) and expressed Cdc42 point mutants that are defective in binding to and activation of its downstream effector molecules PAK and N-WASP were generated, respectively, in CA1 or dentate gyrus (DG) subregion. RESULTS: Cocaine induced upregulation of Cdc42 signaling activity. Cdc42 knockout or mutants blocked cocaine-induced increase in spine plasticity in hippocampal CA1 pyramidal neurons, leading to a decreased conditional place preference (CPP)-associated memories and spatial learning and memory in water maze. Cdc42 knockout or mutants promoted cocaine-induced loss of neurogenesis in DG, leading to a decreased CPP-associated memories and spatial learning and memory in water maze. Furthermore, by using D1R knockout, D2R knockout, and D2R/Cdc42 double knockout mice, we found that D2R, but not D1R, regulated Cdc42 signaling in cocaine-induced neural plasticity and behavioral changes. CONCLUSIONS: Cdc42 acts downstream of D2R in the hippocampus and plays an important role in cocaine-induced neural plasticity through N-WASP and PAK-LIMK-Cofilin, and Cdc42 signaling pathway correlatively links specific brain regions (CA1, dentate gyrus) to cocaine-induced CPP behavior.

Cdc42 regulates extracellular matrix remodeling in three dimensions.

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions.