Summary.  Background: Agonist‐induced inside‐out signaling activates platelet integrin αIIbβ3, rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside‐in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside‐in signaling through αIIbβ3 regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating αIIbβ3 function. Objective: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. Methods and Results: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl3‐induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside‐out signaling appears normal as ADP‐, collagen‐ and PAR4 peptide‐induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside‐in signaling‐dependent tyrosine phosphorylation of the integrin β3 subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. Conclusion: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.

Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer subtype and due to the lack of hormone receptors and HER2 expression, TNBC has limited therapeutic options with chemotherapy being the primary choice for systemic therapy. LIM Domain Kinase 2 (LIMK2) is a serine/threonine kinase that plays an important role in the regulation of actin filament dynamics. Here, we show that LIM domain kinase 2 (LIMK2) is overexpressed in TNBC, and short-hairpin RNA (shRNA)-mediated LIMK2 knockdown or its pharmacological inhibition blocks metastatic attributes of TNBC cells. To determine the mechanism by which LIMK2 promotes TNBC metastatic progression, we performed stable isotope labeling by amino acids in cell culture (SILAC) based unbiased large-scale phosphoproteomics analysis. This analysis identified 258 proteins whose phosphorylation was significantly reduced due to LIMK2 inhibition. Among these proteins, we identified SRSF protein kinase 1 (SRPK1), which encodes for a serine/arginine protein kinase specific for the SR (serine/arginine-rich domain) family of splicing factors. We show that LIMK2 inhibition blocked SRPK1 phosphorylation and consequentially its activity. Furthermore, similar to LIMK2, genetic inhibition of SRPK1 by shRNAs or its pharmacological inhibition blocked the metastatic attributes of TNBC cells. Moreover, the pharmacological inhibition of LIMK2 blocked metastatic progression in mice without affecting primary tumor growth. In sum, these results identified LIMK2 as a facilitator of distal TNBC metastasis and a potential target for preventing TNBC metastatic progression.

TP53 mutations are frequently identified in the copy number-high molecular subgroup of endometrial carcinomas (ECs). P53 immunohistochemistry (IHC) is a widely used surrogate marker reflecting the mutational status of TP53, and recent reports have shown ~95% concordance between the two methods in ECs. While these results are promising, studies evaluating the correlation between different p53 IHC staining patterns and comprehensive next-generation sequencing results are still limited. We compared the p53 IHC staining patterns, scored as wild-type, diffuse nuclear overexpression, null/complete absence, and cytoplasmic, to next-generation sequencing results reported by FoundationOneCDx in 43 high-grade ECs: 20 serous ECs, 9 mixed ECs with a serous component, 4 carcinosarcomas with a serous component, and 10 grade 3 endometrioid ECs. The concordance of p53 IHC and TP53 mutation status was 100% (43/43) overall, including 100% (33/33) concordance in tumors with a serous component and 100% (10/10) in endometrioid ECs. Among the 35 tumors with aberrant p53 expression the most commonly observed pattern was diffuse nuclear overexpression seen in 69% (24/35), followed by cytoplasmic staining in 17% (6/35), and complete absence of staining (null) in 14% (5/35) of tumors. Of the 6 tumors with cytoplasmic staining, 4 corresponded to missense mutations within the DNA binding domain (V157F in 2 tumors, and S127P and R280S, in 2 tumor each), while 2 corresponded to nonsense mutations in the tetramerization domain (p.E339*). Our results further support that p53 IHC can serve as an accurate predictor of TP53 alterations in ECs to aid the molecular-based tumor classification and the distinction between tumor histotypes, both of which play an important role in the assessment of clinical prognosis and therapeutic decision making. In addition, our data suggest, that the type and position of TP53 mutation may not directly correlate with the observed p53 IHC pattern in all tumors, and that there may be alternative mechanisms for cytoplasmic localization (other than mutations involving the nuclear localization domain), possibly due to conformational changes or posttranslational modifications of the aberrant p53 protein.