Role of IκB kinase ß in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex.

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKß is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKß regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKß regulates membrane fusion and the remodeling of the CBM complex formation.

P2Y12 antibody inhibits platelet activity and protects against thrombogenesis.

Given that platelet hyperactivity is known to give rise to thrombotic disorders, new and/or novel antiplatelet therapies are constantly being developed to add to, or to complement the current arsenal of agents. To this end, adenosine diphosphate (ADP) is an important platelet activator that acts by binding to the G-protein coupled P2Y1 and P2Y12 receptors. Although the contribution of the P2Y12 receptor to the genesis of thrombosis is well established, the parenteral arsenal of drugs targeting this receptor in clinical use is limited to cangrelor. In this study, we investigated the potential antiplatelet activity of an antibody targeting the ligand-binding domain of the P2Y12 receptor (abbreviated P2Y12Ab). Our in vitro studies revealed that the P2Y12Ab could effectively inhibit aggregation induced by ADP, as well as that triggered by the thromboxane receptor agonist U46619. Additionally, using FACS analysis, we observed reduced P-selectin, phosphatidylserine exposure and integrin activation in the presence of P2Y12Ab. As for its in vivo effects, the P2Y12Ab also demonstrated protection against thrombus formation; albeit this was accompanied with a bleeding diathesis (longer bleeding time). Notably, this inhibitory profile is consistent with that observed with oral anti-P2Y12 agents. Collectively, our findings demonstrate that the P2Y12Ab functionally blocks platelet activity in vitro and in vivo, and support the notion that it can be purposed as a parenteral antiplatelet agent, to be used in conjunction with and/or as a complement to current antiplatelet therapies.

Sensitization to Ionizing Radiation by MEK Inhibition Is Dependent on SNAI2 in Fusion-Negative Rhabdomyosarcoma.

In fusion-negative rhabdomyosarcoma (FN-RMS), a pediatric malignancy with skeletal muscle characteristics, >90% of high-risk patients have mutations that activate the RAS/MEK signaling pathway. We recently discovered that SNAI2, in addition to blocking myogenic differentiation downstream of MEK signaling in FN-RMS, represses proapoptotic BIM expression to protect RMS tumors from ionizing radiation (IR). As clinically relevant concentrations of the MEK inhibitor trametinib elicit poor responses in preclinical xenograft models, we investigated the utility of low-dose trametinib in combination with IR for the treatment of RAS-mutant FN-RMS. We hypothesized that trametinib would sensitize FN-RMS to IR through its downregulation of SNAI2 expression. While we observed little to no difference in myogenic differentiation or cell survival with trametinib treatment alone, robust differentiation and reduced survival were observed after IR. In addition, IR-induced apoptosis was significantly increased in FN-RMS cells treated concurrently with trametinib, as was increased BIM expression. SNAI2's role in these processes was established using overexpression rescue experiments, where overexpression of SNAI2 prevented IR-induced myogenic differentiation and apoptosis. Moreover, combining MEK inhibitor with IR resulted in complete tumor regression and a 2- to 4-week delay in event-free survival (EFS) in preclinical xenograft and patient-derived xenograft models. Our findings demonstrate that the combination of MEK inhibition and IR results in robust differentiation and apoptosis, due to the reduction of SNAI2, which leads to extended EFS in FN-RMS. SNAI2 thus is a potential biomarker of IR insensitivity and target for future therapies to sensitize aggressive sarcomas to IR.

Zebrafish Tumor Graft Transplantation to Grow Tumors In Vivo That Engraft Poorly as Single Cell Suspensions.

Angiosarcoma is a clinically aggressive tumor with a high rate of mortality. It can arise in vascular or lymphatic tissues, involve any part of the body, and aggressively spread locally or metastasize. Angiosarcomas spontaneously develop in the tp53 deleted (tp53del/del) zebrafish mutant. However, established protocols for tumor dissection and transplantation of single cell suspensions of angiosarcoma tumors result in inferior implantation rates. To resolve these complications, we developed a new tumor grafting technique for engraftment of angiosarcoma and similar tumors in zebrafish, which maintains the tumor microenvironment and has superior rates of engraftment.

Interaction between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in Fusion Negative Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis.

SNAI2-Mediated Repression of BIM Protects Rhabdomyosarcoma from Ionizing Radiation.

Ionizing radiation (IR) and chemotherapy are mainstays of treatment for patients with rhabdomyosarcoma, yet the molecular mechanisms that underlie the success or failure of radiotherapy remain unclear. The transcriptional repressor SNAI2 was previously identified as a key regulator of IR sensitivity in normal and malignant stem cells through its repression of the proapoptotic BH3-only gene PUMA/BBC3. Here, we demonstrate a clear correlation between SNAI2 expression levels and radiosensitivity across multiple rhabdomyosarcoma cell lines. Modulating SNAI2 levels in rhabdomyosarcoma cells through its overexpression or knockdown altered radiosensitivity in vitro and in vivo. SNAI2 expression reliably promoted overall cell growth and inhibited mitochondrial apoptosis following exposure to IR, with either variable or minimal effects on differentiation and senescence, respectively. Importantly, SNAI2 knockdown increased expression of the proapoptotic BH3-only gene BIM, and chromatin immunoprecipitation sequencing experiments established that SNAI2 is a direct repressor of BIM/BCL2L11. Because the p53 pathway is nonfunctional in the rhabdomyosarcoma cells used in this study, we have identified a new, p53-independent SNAI2/BIM signaling axis that could potentially predict clinical responses to IR treatment and be exploited to improve rhabdomyosarcoma therapy. SIGNIFICANCE: SNAI2 is identified as a major regulator of radiation-induced apoptosis in rhabdomyosarcoma through previously unknown mechanisms independent of p53.

RGS10 Negatively Regulates Platelet Activation and Thrombogenesis.

Regulators of G protein signaling (RGS) proteins act as GTPase activating proteins to negatively regulate G protein-coupled receptor (GPCR) signaling. Although several RGS proteins including RGS2, RGS16, RGS10, and RGS18 are expressed in human and mouse platelets, the respective unique function(s) of each have not been fully delineated. RGS10 is a member of the D/R12 subfamily of RGS proteins and is expressed in microglia, macrophages, megakaryocytes, and platelets. We used a genetic approach to examine the role(s) of RGS10 in platelet activation in vitro and hemostasis and thrombosis in vivo. GPCR-induced aggregation, secretion, and integrin activation was much more pronounced in platelets from Rgs10-/- mice relative to wild type (WT). Accordingly, these mice had markedly reduced bleeding times and were more susceptible to vascular injury-associated thrombus formation than control mice. These findings suggest a unique, non-redundant role of RGS10 in modulating the hemostatic and thrombotic functions of platelets in mice. RGS10 thus represents a potential therapeutic target to control platelet activity and/or hypercoagulable states.