A target discovery pipeline identified ILT3 as a target for immunotherapy of multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.

Loss of the volume-regulated anion channel components LRRC8A and LRRC8D limits platinum drug efficacy.

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response, e.g. due to dysfunctional BRCA1. Despite this knowledge, we are still missing helpful markers to predict Pt response in the clinic. We have previously shown that volume-regulated anion channels, containing the subunits LRRC8A and LRRC8D, promote the uptake of cisplatin and carboplatin in BRCA1-proficient cell lines. Here, we show that the loss of LRRC8A or LRRC8D significantly reduces the uptake of cis- and carboplatin in BRCA1;p53-deficient mouse mammary tumor cells. This results in reduced DNA damage and in vivo drug resistance. In contrast to Lrrc8a, the deletion of the Lrrc8d gene does not affect the viability and fertility of mice. Interestingly, Lrrc8d-/- mice tolerate a two-fold cisplatin maximum-tolerable dose. This allowed us to establish a mouse model for intensified Pt-based chemotherapy, and we found that an increased cisplatin dose eradicates BRCA1;p53-deficient tumors, whereas eradication is not possible in WT mice. Moreover, we show that decreased expression of LRRC8A/D in head and neck squamous cell carcinoma patients, who are treated with a Pt-based chemoradiotherapy, leads to decreased overall survival of the patients. In particular, high cumulative cisplatin dose treatments lost their efficacy in patients with a low LRRC8A/D expression in their cancers. Our data therefore suggest that LRRC8A and LRRC8D should be included in a prospective trial to predict the success of intensified cis- or car-boplatin-based chemotherapy.

LRRC8A:C/E Heteromeric Channels Are Ubiquitous Transporters of cGAMP.

Extracellular 2'3'-cyclic-GMP-AMP (cGAMP) is an immunotransmitter exported by diseased cells and imported into host cells to activate the innate immune STING pathway. We previously identified SLC19A1 as a cGAMP importer, but its use across human cell lines is limited. Here, we identify LRRC8A heteromeric channels, better known as volume-regulated anion channels (VRAC), as widely expressed cGAMP transporters. LRRC8A forms complexes with LRRC8C and/or LRRC8E, depending on their expression levels, to transport cGAMP and other 2'3'-cyclic dinucleotides. In contrast, LRRC8D inhibits cGAMP transport. We demonstrate that cGAMP is effluxed or influxed via LRRC8 channels, as dictated by the cGAMP electrochemical gradient. Activation of LRRC8A channels, which can occur under diverse stresses, strongly potentiates cGAMP transport. We identify activator sphingosine 1-phosphate and inhibitor DCPIB as chemical tools to manipulate channel-mediated cGAMP transport. Finally, LRRC8A channels are key cGAMP transporters in resting primary human vasculature cells and universal human cGAMP transporters when activated.

Determination of the mutational landscape in Taiwanese patients with papillary thyroid cancer by whole-exome sequencing.

Among women in Taiwan, thyroid cancer is the fifth most common malignant neoplasia. However, genomic profiling of papillary thyroid cancer (PTC) cases from Taiwan has not been attempted previously. We used whole-exome sequencing to identify mutations in a cohort of 19 PTC patients. Sequencing was performed using the Illumina system; Sanger sequencing was used to validate all identified mutations. We identified new somatic mutations in APC, DICER1, LRRC8D and NDRG1. We also found somatic mutations in ARID5A, CREB3L2, MDM4, PPP2R5A and TFPT; mutations in these genes had been found previously in other tumors, but had not been described previously in PTC. We also investigated the pathway deregulation in BRAF-mutated PTC compared with wild-type BRAF PTC. In checking our gene mutations against The Cancer Genome Atlas (TCGA) database, we identified aberrations in one pathway that are specific to BRAF-mutated PTC: maturity-onset diabetes of the young. In addition, the caffeine metabolism pathway showed aberrations that are specific to wild-type BRAF PTC. For this study, we performed a comprehensive exome-wide analysis of the mutational spectra of Taiwanese patients with PTC. We identified novel genes that are potentially associated with PTC tumorigenesis, as well as aberrations in pathways that led to the distinct pathogeneses of BRAF-mutated PTC and wild-type BRAF PTC, which may provide a new target for PTC therapy.

The protein synthesis inhibitor blasticidin s enters mammalian cells via leucine-rich repeat-containing protein 8D.

Leucine-rich repeat-containing 8 (LRRC8) proteins have been identified as putative receptors involved in lymphocyte development and adipocyte differentiation. They remain poorly characterized, and no specific function has been assigned to them. There is no consensus on how this family of proteins might function because homology searches suggest that members of the LRRC8 family act not as plasma membrane receptors, but rather as channels that mediate cell-cell signaling. Here we provide experimental evidence that supports a role for LRRC8s in the transport of small molecules. We show that LRRC8D is a mammalian protein required for the import of the antibiotic blasticidin S. We characterize localization and topology of LRRC8A and LRRC8D and demonstrate that LRRC8D interacts with LRRC8A, LRRC8B, and LRRC8C. Given the suggested involvement in solute transport, our results support a model in which LRRC8s form one or more complexes that may mediate cell-cell communication by transporting small solutes.