Integrins and ERp57 Coordinate to Regulate Cell Surface Calreticulin in Immunogenic Cell Death.

Therapy-induced presentation of cell surface calreticulin (CRT) is a pro-phagocytic immunogen beneficial for invoking anti-tumor immunity. Here, we characterized the roles of ERp57 and α-integrins as CRT-interacting proteins that coordinately regulate CRT translocation from the ER to the surface during immunogenic cell death. Using T-lymphoblasts as a genetic cell model, we found that drug-induced surface CRT is dependent on ERp57, while drug-induced surface ERp57 is independent of CRT. Differential subcellular immunostaining assays revealed that ERp57-/- cells have minimal cytosolic CRT, indicating that ERp57 is indispensable for extra-ER accumulation of CRT. Stimulation of integrin activity, with either cell adhesion or molecular agonists, resulted in decreased drug-induced surface CRT and ERp57 levels. Similarly, surface CRT and ERp57 was reduced in cells expressing GFFKR, a conserved α-integrin cytosolic motif that binds CRT. Drug-induced surface ERp57 levels were consistently higher in CRT-/- cells, suggesting integrin inhibition of surface ERp57 is an indirect consequence of α-integrin binding to CRT within the CRT-ERp57 complex. Furthermore, ß1-/- cells with reduced expression of multiple α-integrins, exhibit enhanced levels of drug-induced surface CRT and ERp57. Our findings highlight the coordinate involvement of plasma membrane integrins as inhibitors, and ERp57 originating from the ER as promoters, of CRT translocation from the ER to the cell surface.

CD47-ligation induced cell death in T-acute lymphoblastic leukemia.

CD47 is a cell-surface marker well recognized for its anti-phagocytic functions. As such, an emerging avenue for targeted cancer therapies involves neutralizing the anti-phagocytic function using monoclonal antibodies (mAbs) to enhance tumour cell immunogenicity. A lesser known consequence of CD47 receptor ligation is the direct induction of tumour cell death. While several mAbs and their derivatives with this property have been studied, the best characterized is the commercially available mAb B6H12, which requires immobilization for induction of cell death. Here, we describe a commercially available mAb, CC2C6, which induces T-cell acute lymphoblastic leukemia (ALL) cell death in soluble form. Soluble CC2C6 induces CD47-dependent cell death in a manner consistent with immobilized B6H12, which is characterized by mitochondrial deficiencies but is independent of caspase activation. Titration studies indicated that CC2C6 shares a common CD47-epitope with B6H12. Importantly, CC2C6 retains the anti-phagocytic neutralizing function, thus possessing dual anti-tumour properties. Although CD47-ligation induced cell death occurs in a caspase-independent manner, CC2C6 was found to stimulate increases in Mcl-1 and NOXA levels, two Bcl-2 family proteins that govern the intrinsic apoptosis pathway. Further analysis revealed that the ratio of Mcl-1:NOXA were minimally altered for cells treated with CC2C6, in comparison to cells treated with agents that induced caspase-dependent apoptosis which alter this ratio in favour of NOXA. Finally, we found that CC2C6 can synergize with low dose chemotherapeutic agents that induce classical apoptosis, giving rise to the possibility of an effective combination treatment with reduced long-term sequelae associated with high-dose chemotherapies in childhood ALL.

Tumor Variant Identification That Accounts for the Unique Molecular Landscape of Pediatric Malignancies.

Precision oncology trials for pediatric cancers require rapid and accurate detection of genetic alterations. Tumor variant identification should interrogate the distinctive driver genes and more frequent copy number variants and gene fusions that are characteristics of pediatric tumors. Here, we evaluate tumor variant identification using whole genome sequencing (n = 12 samples) and two amplification-based next-generation sequencing assays (n = 28 samples), including one assay designed to rapidly assess common diagnostic, prognostic, and therapeutic biomarkers found in pediatric tumors. Variant identification by the three modalities was comparable when filtered for 151 pediatric driver genes. Across the 28 samples, the pediatric cancer-focused assay detected more tumor variants per sample (two-sided, P < .05), which improved the identification of potentially druggable events and matched pathway inhibitors. Overall, our data indicate that an assay designed to evaluate pediatric cancer-specific variants, including gene fusions, may improve the detection of target-agent pairs for precision oncology.

The membrane-proximal KXGFFKR motif of α-integrin mediates chemoresistance.

Cell adhesion-mediated drug resistance contributes to minimal residual disease and relapse in hematological malignancies. Here, we show that adhesion of Jurkat T-acute lymphoblastic leukemia cells to substrates engaging α4ß1-integrin or α5ß1-integrin promotes chemoresistance to doxorubicin-induced apoptosis. Reconstituted expression of α4δ, a truncated α4-integrin with KXGFFKR as the cytoplasmic motif, in α4-deficient cells promoted chemoresistance to doxorubicin in a manner independent of α4-mediated adhesion. The adhesion-independent chemoresistance did not require ß1-integrin as the heterodimeric pair, since expression of Tacδ, a monomeric nonintegrin transmembrane protein fused to the juxtamembrane KXGFFKR, was sufficient to reproduce the phenomenon. The requirement for integrin-mediated adhesion in stimulation of Akt phosphorylation and activation was bypassed for cells expressing α4δ and Tacδ. Cells expressing α4δ and Tacδ exhibited a high influx of extracellular Ca(2+), and inhibition of Ca(2+) channels with verapamil attenuated the adhesion-independent chemoresistance. Tacδ cells also exhibited greater rates of drug efflux. α4δ and Tacδ interacted with the Ca(2+)-binding protein calreticulin, in a manner dependent on the KXGFFKR motif. Adhesion-mediated engagement of α4-integrins promoted an increased calreticulin-α4 association and greater influx of extracellular Ca(2+) than in nonadherent cells. The α-integrin KXGFFKR motif is involved in adhesion-mediated control of chemoresistance in T cells.

Two modes of integrin activation form a binary molecular switch in adhesion maturation.

Talin-mediated integrin activation drives integrin-based adhesions. Here we examine the roles of two proteins that induce talin-integrin interactions--vinculin and Rap1-GTP-interacting adaptor molecule (RIAM)--in the formation and maturation of integrin-based adhesions. RIAM-containing adhesions are primarily in the lamellipodium; RIAM is subsequently reduced in mature focal adhesions due to direct competition with vinculin for talin-binding sites. We show that vinculin binding to talin induces Rap1-independent association of talin with integrins and resulting integrin activation, in sharp contrast to Rap1-dependent RIAM-induced activation. Vinculin stabilizes adhesions, increasing their ability to transmit force, whereas RIAM played a critical role in lamellipodial protrusion. Thus displacement of RIAM by vinculin acts as a molecular switch that mediates the transition of integrin-based adhesions from drivers of lamellipodial protrusion to stable, force-bearing adhesions. Consequently changes in the abundance of two multiprotein modules within maturing adhesions, one regulated by Rap1 and one by tension, result in the temporal evolution of adhesion functions.

Genetic variation within the hypothalamus-pituitary-ovarian axis in women with recurrent miscarriage.

BACKGROUND: Recurrent miscarriage affects 1-2% of couples trying to conceive, and is idiopathic in nearly half. Female fertility is controlled by the hypothalamus-pituitary-ovarian (HPO) axis and we hypothesize that genetic polymorphisms affecting the function of genes involved in regulating the HPO axis will be associated with recurrent miscarriage. METHODS: Whole peripheral blood DNA from 227 women with recurrent miscarriage and 130 control women was obtained for this study. Using the Sequenom iPlex assay for fragment analysis, 31 single-nucleotide polymorphisms (SNPs) and 4 short tandem repeat (STR) polymorphisms in 20 candidate genes were evaluated for genetic association with recurrent miscarriage. RESULTS: Several candidate associations were identified with an uncorrected P-value of 0.05. Genotype distribution at an SNP (rs37389) in the prolactin receptor gene (P = 0.03), and allele distributions at an SNP (rs41423247) in the glucocorticoid receptor gene (P = 0.04) and an STR polymorphism in the estrogen receptor ß gene (P = 0.03) were associated with recurrent miscarriage. The T allele of an SNP (rs2033962) within the activin receptor type 1 gene (ACVR1) was associated with increased number of miscarriages in an additive manner (P = 0.02). These candidate associations were not statistically significant after correcting for multiple analyses. CONCLUSIONS: Candidate associations were identified between recurrent miscarriage and genetic variation within ESR2, PRLR, GCCR and ACVR1 genes. Independent confirmation of these results is needed, as limitations of this study include the heterogeneous etiology of recurrent miscarriage, limited sample size, partial availability of reproductive history of the control group and investigation of only a subset of the genetic variation within each gene.