T 5601640

T56-LIMKi is a selective inhibitor of LIMK2; inhibits the growth of Panc-1 cells with an IC50 of 35.2 μM.

In Vitro:T56-LIMKi efficiently inhibits the growth of ST88-14, U87, Panc-1 cells, A549 lung cancer cells with IC50 values of 18.3, 7.4, 35.2 and 90 μM, respectively. T56-LIMKi decreases phosphorylated cofilin (p-cofilin) levels and thus inhibits growth of several cancerous cell lines, including those of pancreatic cancer, glioma and schwannoma[1]. It blocks the phosphorylation of cofilin which leads to actin severance and inhibition of tumor cell migration, tumor cell growth, and anchorage-independent colony formation in soft agar. T56-LIMKi (10-50 μM) reduces p-cofilin in a dose-dependent manner in NF1−/−MEFs with an IC50 of 30 μM. Notably, the inhibitor does not affect the amounts of total cofil. 50μM T56-LIMKi causes a statistically significant reduction in the number of cells exhibiting stress fibers[2].

In Vivo:T56-LIMKi can induce inhibition of cofilin phosphorylation and Panc-1 tumor shrinkage in vivo. Mice treated with T56-LIMKi (60 mg/kg) shows a significant decrease in tumor volume compared to control[1].

References:
[1]. Rak R, et al. Novel LIMK2 Inhibitor Blocks Panc-1 Tumor Growth in a mouse xenograft model. Oncoscience. 2014 Jan 1;1(1):39-48. eCollection 2014. [2]. Mashiach-Farkash E, et al. Computer-based identification of a novel LIMK1/2 inhibitor that synergizes with salirasib to destabilize the actin cytoskeleton. Oncotarget. 2012 Jun;3(6):629-39.

Local Inflammatory Cues Regulate Differentiation and Persistence of CD8(+) Tissue-Resident Memory T Cells.[Pubmed:28380351]

Cell Rep. 2017 Apr 4;19(1):114-124.

Many pathogens initiate infection at mucosal surfaces, and tissue-resident memory T (Trm) cells play an important role in protective immunity, yet the tissue-specific signals that regulate Trm differentiation are poorly defined. During Yersinia infection, CD8(+) T cell recruitment to areas of inflammation within the intestine is required for differentiation of the CD103(-)CD69(+) Trm subset. Intestinal proinflammatory microenvironments have elevated interferon (IFN)-beta and interleukin-12 (IL-12), which regulated Trm markers, including CD103. Type I interferon-receptor- or IL-12-receptor-deficient T cells functioned similarly to wild-type (WT) cells during infection; however, the inability of T cells to respond to inflammation resulted in defective differentiation of CD103(-)CD69(+) Trm cells and reduced Trm persistence. Intestinal macrophages were the main producers of IFN-beta and IL-12 during infection, and deletion of CCR2(+) IL-12-producing cells reduced the size of the CD103(-) Trm population. These data indicate that intestinal inflammation drives phenotypic diversity and abundance of Trm cells for optimal tissue-specific immunity.

Clinical and prognostic significance of aberrant T-cell marker expression in 225 cases of de novo diffuse large B-cell lymphoma and 276 cases of other B-cell lymphomas.[Pubmed:28380441]

Oncotarget. 2017 May 16;8(20):33487-33500.

Expression of T-cell markers, generally investigated for immunophenotyping of T-cell lymphomas, is also observed in several types of B-cell lymphomas, including diffuse large B-cell lymphoma (DLBCL). We previously reported that CD5 expression in DLBCL is an inferior prognostic factor in the era of rituximab. However, data regarding the frequencies, histological relevance, and prognostic importance of T-cell markers other than CD5 are currently unavailable. In the present study, we comprehensively evaluated the expression of T-cell markers (CD2, CD3, CD4, CD5, CD7, and CD8) in 501 B-cell lymphomas, including 225 DLBCLs, by flow cytometry and subsequent immunohistochemistry. T-cell markers other than CD5, such as CD2, CD4, CD7, and CD8, were expressed in 27 (5%) patients, and notably, all of these cases were classified as large B-cell lymphoma subtypes: 25 DLBCLs and 2 intravascular large B-cell lymphomas. CD5 and other T-cell markers were expressed in 15% (31/225) and 10% (25/225) of DLBCL cases, respectively. Five of them co-expressed CD5 and other T-cell markers. Retrospectively analyzing the prognostic relevance of T-cell markers in 169 patients with primary DLBCL treated with rituximab-based chemotherapy, we showed that only CD5 was a strong predictor of poor survival. This study provides information about the occurrence of T-cell markers other than CD5 in B-cell lymphomas, their frequent histological subtypes, and their prognostic significance in DLBCL. CD5 was reconfirmed as a negative prognostic marker in DLBCL patients receiving rituximab-inclusive chemotherapy, whereas T-cell markers other than CD5 were found to have no impact on clinicopathological and survival analyses.

Hemodialysis Patients Display a Declined Proportion of Th2 and Regulatory T Cells in Parallel with a High Interferon-gamma Profile.[Pubmed:28380480]

Nephron. 2017;136(3):254-260.

BACKGROUND: A high prevalence of cardiovascular diseases (CVDs) and infections in patients with chronic kidney disease (CKD) arises partly due to a high inflammatory state and aberrations in immune cells function. Following in vitro stimulation of leukocytes with different T-cell mitogens, we observed a lower level of interleukin (IL)-2 and IL-10 production in CKD patients. To gain more knowledge as to whether this is the result of an alteration in T-cell function, we investigated the T-cell subsets profile and cytokine production in hemodialysis patients. METHODS: CD4+ cells were isolated from whole blood of 10 hemodialysis patients and 10 age- and gender-matched healthy controls. Following in vitro stimulation with an antigen-independent T-cell mitogen, Th1, Th2, and regulatory T (Treg) cell subsets were analyzed by flow cytometry through the expression of specific transcription factors. The levels of cytokines, interferon (IFN)-gamma, IL-4, and IL-10 were analyzed by enzyme-linked immunosorbent assay in the supernatants. RESULTS: The proportion of CD4+CD25+FOXP3+ (Treg) and CD4+GATA3+ (Th2) cells was significantly lower in patients compared to healthy controls, while the proportion of CD4+T-bet+ (Th1) cells was similar. Moreover, levels of IL-4 were significantly lower in supernatants from patients, while IFN-gamma levels were higher. IL-10 levels did not differ compared to those of the healthy controls. CONCLUSIONS: Our findings indicate a diminished anti-inflammatory Treg, and Th2 cell profile in hemodialysis patients, accompanied by a high pro-inflammatory IFN-gamma profile. Since this profile is characterized in CVDs, we propose that an imbalance between the inflammatory and anti-inflammatory responses may contribute to the pathogenesis of CVD in advanced CKD.

Wnt5a and CCL25 promote adult T-cell acute lymphoblastic leukemia cell migration, invasion and metastasis.[Pubmed:28380463]

Oncotarget. 2017 Jun 13;8(24):39033-39047.

Adult T-cell acute lymphoblastic leukemia (T-ALL) is a refractory leukemia. We previously showed that CCL25/CCR9 promotes T-ALL metastasis. In the present study, we assessed the effects of CCL25 on Wnt expression and the effects of Wnt5a and CCL25 on PI3K/Akt and RhoA activation. Transwell assays and mouse xenograft experiments were utilized to assess the effects of Wnt5a and CCL25 on MOLT4 cell invasion, migration and metastasis. The effects of Wnt5a on MOLT4 cell actin polarization and pseudopodium formation were examined using laser scanning confocal microscopy and scanning electron microscopy. CCL25 induced Wnt5a expression in MOLT4 cells by promoting protein kinase C (PKC) expression and activation. Wnt5a promoted MOLT4 cell migration, invasion, actin polarization, and lamellipodium and filopodia formation via PI3K/Akt-RhoA pathway activation. These effects were rescued by PI3K/Akt or RhoA knockdown or inhibition. Additionally, Wnt5a in cooperation with CCL25 promoted MOLT4 cell mouse liver metastasis and stimulated RhoA activation. These results show that CCL25/CCR9 upregulates Wnt5a by promoting PKC expression and activation in MOLT4 cells. This in turn promotes cell migration and invasion via PI3K/Akt-RhoA signaling, enhancing cell polarization and pseudopodium formation. These findings indicate that the PI3K/Akt-RhoA pathway is likely responsible for Wnt5a-induced adult T-ALL cell migration and invasion.

Novel LIMK2 Inhibitor Blocks Panc-1 Tumor Growth in a mouse xenograft model.[Pubmed:25593987]

Oncoscience. 2014 Jan 1;1(1):39-48.

LIM kinases (LIMKs) are important cell cytoskeleton regulators that play a prominent role in cancer manifestation and neuronal diseases. The LIMK family consists of two homologues, LIMK1 and LIMK2, which differ from one another in expression profile, intercellular localization, and function. The main substrate of LIMK is cofilin, a member of the actin-depolymerizing factor (ADF) protein family. When phosphorylated by LIMK, cofilin is inactive. LIMKs play a contributory role in several neurodevelopmental disorders and in cancer growth and metastasis. We recently reported the development and validation of a novel LIMK inhibitor, referred to here as T56-LIMKi, using a combination of computational methods and classical biochemistry techniques. Here we report that T56-LIMKi inhibits LIMK2 with high specificity, and shows little or no cross-reactivity with LIMK1. We found that T56-LIMKi decreases phosphorylated cofilin (p-cofilin) levels and thus inhibits growth of several cancerous cell lines, including those of pancreatic cancer, glioma and schwannoma. Because the most promising in-vitro effect of T56-LIMKi was observed in the pancreatic cancer cell line Panc-1, we tested the inhibitor on a nude mouse Panc-1 xenograft model. T56-LIMKi reduced tumor size and p-cofilin levels in the Panc-1 tumors, leading us to propose T56-LIMKi as a candidate drug for cancer therapy.

Computer-based identification of a novel LIMK1/2 inhibitor that synergizes with salirasib to destabilize the actin cytoskeleton.[Pubmed:22776759]

Oncotarget. 2012 Jun;3(6):629-39.

Neurofibromin regulates cell motility via three distinct GTPase pathways acting through two different domains, the Ras GTPase-activating protein-related domain (GRD) and the pre-GRD domain. First, the GRD domain inhibits Ras-dependent changes in cell motility through the mitogen activated protein cascade. Second, it also regulates Rho-dependent (Ras-independent) changes by activating LIM kinase 2 (LIMK2), an enzyme that phosphorylates and inactivates cofilin (an actin-depolymerizing factor). Third, the pre-GRD domain acts through the Rac1 GTPase, that activate the P21 activated kinase 1 (PAK1)-LIMK1-cofilin pathway. We employed molecular modeling to identify a novel inhibitor of LIMK1/2. The active sites of an ephrin-A receptor (EphA3) and LIMK2 showed marked similarity (60%). On testing a known inhibitor of EphA3, we found that it fits to the LIMK1/2-ATP binding site and to the latter's substrate-binding pockets. We identified a similar compound, T56-LIMKi, and found that it inhibits LIMK1/2 kinase activities. It blocked the phosphorylation of cofilin which led to actin severance and inhibition of tumor cell migration, tumor cell growth, and anchorage-independent colony formation in soft agar. Because modulation of LIMK by neurofibromin is not affected by the Ras inhibitor Salirasib, we examined the combined effect of Salirasib and T56-LIMKi each of which can affect cell motility by a distinct pathway. We found that their combined action on cell proliferation and stress-fiber formation in neurofibromin-deficient cells was synergistic. We suggest that this drug combination may be developed for treatment of neurofibromatosis and cancer.