Synergistic melanoma cell death mediated by inhibition of both MCL1 and BCL2 in high-risk tumors driven by NF1/PTEN loss

Melanomas driven by loss of the NF1 tumor suppressor have a high risk of treatment failure and effective therapies have not been developed. Here we show that loss-of-function mutations of nf1 and pten result in aggressive melanomas in zebrafish, representing the first animal model of NF1-mutant melanomas harboring PTEN loss. MEK or PI3K inhibitors show little activity when given alone due to cross-talk between the pathways, and high toxicity when given together. The mTOR inhibitors, sirolimus, everolimus, and temsirolimus, were the most active single agents tested, potently induced tumor-suppressive autophagy, but not apoptosis. Because addition of the BCL2 inhibitor venetoclax resulted in compensatory upregulation of MCL1, we established a three-drug combination composed of sirolimus, venetoclax, and the MCL1 inhibitor S63845. This well-tolerated drug combination potently and synergistically induces apoptosis in both zebrafish and human NF1/PTEN-deficient melanoma cells, providing preclinical evidence justifying an early-stage clinical trial in patients with NF1/PTEN-deficient melanoma

Antileukemic Activity of Nuclear Export Inhibitors that Spare Normal Hematopoietic Cells

Drugs that target the chief mediator of nuclear export, chromosome region maintenance 1 protein (CRM1) have potential as therapeutics for leukemia, but existing CRM1 inhibitors show variable potencies and a broad range of cytotoxic effects. Here, we report the structural analysis and antileukemic activity of a new generation of small-molecule inhibitors of CRM1. Designated selective inhibitors of nuclear export (SINE), these compounds were developed using molecular modeling to screen a small virtual library of compounds against the nuclear export signal (NES) groove of CRM1. The 2.2-Å crystal structure of the CRM1-Ran-RanBP1 complex bound to KPT-251, a representative molecule of this class of inhibitors, shows that the drug occupies part of the groove in CRM1 that is usually occupied by the NES, but penetrates much deeper into the groove and blocks CRM1-directed protein export. SINE inhibitors exhibit potent antileukemic activity, inducing apoptosis at nanomolar concentrations in a panel of 14 human acute myeloid leukemia (AML) cell lines representing different molecular subtypes of the disease. When administered orally to immunodeficient mice engrafted with human AML cells, KPT-251 had potent antileukemic activity with negligible toxicity to normal hematopoietic cells. Thus, KPT-SINE CRM1 antagonists represent a novel class of drugs that warrant further testing in AML patients

Immunohistochemical Detection of MYC-driven Diffuse Large B-Cell Lymphomas

Diffuse large B cell lymphoma (DLBCL) is a clinically and genetically heterogeneous disease. A small subset of DLBCLs has translocations involving the MYC locus and an additional group has a molecular signature resembling Burkitt lymphoma (mBL). Presently, identification of such cases by morphology is unreliable and relies on cytogenetic or complex molecular methods such as gene transcriptional profiling. Herein, we describe an immunohistochemical (IHC) method for identifying DLBCLs with increased MYC protein expression. We tested 77 cases of DLBCL and identified 15 cases with high MYC protein expression (nuclear staining in >50% of tumor cells). All MYC translocation positive cases had increased MYC protein expression by this IHC assay. In addition, gene set enrichment analysis (GSEA) of the DLBCL transcriptional profiles revealed that tumors with increased MYC protein expression (regardless of underlying MYC translocation status) had coordinate upregulation of MYC target genes, providing molecular confirmation of the IHC results. We then generated a molecular classifier derived from the MYC IHC results in our cases and employed it to successfully classify mBLs from two previously reported independent case series, providing additional confirmation that the MYC IHC results identify clinically important subsets of DLBCLs. Lastly, we found that DLBCLs with high MYC protein expression had inferior overall survival when treated with R-CHOP. In conclusion, the IHC method described herein can be used to readily identify the biologically and clinically distinct cases of MYC-driven DLBCL, which represent a clinically significant subset of DLBCL cases due to their inferior overall survival

Whole Exome Sequencing of Patients with Steroid-Resistant Nephrotic Syndrome

BACKGROUND AND OBJECTIVES: Steroid-resistant nephrotic syndrome overwhelmingly progresses to ESRD. More than 30 monogenic genes have been identified to cause steroid-resistant nephrotic syndrome. We previously detected causative mutations using targeted panel sequencing in 30% of patients with steroid-resistant nephrotic syndrome. Panel sequencing has a number of limitations when compared with whole exome sequencing. We employed whole exome sequencing to detect monogenic causes of steroid-resistant nephrotic syndrome in an international cohort of 300 families. DESIGN, SETTING, PARTIIPANTS AND MEASUREMENTS: Three hundred thirty-five individuals with steroid-resistant nephrotic syndrome from 300 families were recruited from April of 1998 to June of 2016. Age of onset was restricted to <25 years of age. Exome data were evaluated for 33 known monogenic steroid-resistant nephrotic syndrome genes. RESULTS: In 74 of 300 families (25%), we identified a causative mutation in one of 20 genes known to cause steroid-resistant nephrotic syndrome. In 11 families (3.7%), we detected a mutation in a gene that causes a phenocopy of steroid-resistant nephrotic syndrome. This is consistent with our previously published identification of mutations using a panel approach. We detected a causative mutation in a known steroid-resistant nephrotic syndrome gene in 38% of consanguineous families and in 13% of nonconsanguineous families, and 48% of children with congenital nephrotic syndrome. A total of 68 different mutations were detected in 20 of 33 steroid-resistant nephrotic syndrome genes. Fifteen of these mutations were novel. NPHS1, PLCE1, NPHS2, and SMARCAL1 were the most common genes in which we detected a mutation. In another 28% of families, we detected mutations in one or more candidate genes for steroid-resistant nephrotic syndrome. CONCLUSIONS: Whole exome sequencing is a sensitive approach toward diagnosis of monogenic causes of steroid-resistant nephrotic syndrome. A molecular genetic diagnosis of steroid-resistant nephrotic syndrome may have important consequences for the management of treatment and kidney transplantation in steroid-resistant nephrotic syndrome

Targeting CD22 Reprograms B-Cells and Reverses Autoimmune Diabetes

OBJECTIVES—To investigate a B-cell–depleting strategy to reverse diabetes in naïve NOD mice

B7-H1-Deficiency Enhances the Potential of Tolerogenic Dendritic Cells by Activating CD1d-Restricted Type II NKT Cells

Background: Dendritic cells (DC) can act tolerogenic at a semi-mature stage by induction of protective CD4+ T cell and NKT cell responses. Methodology/Principal Findings: Here we studied the role of the co-inhibitory molecule B7-H1 (PD-L1, CD274) on semimature DC that were generated from bone marrow (BM) cells of B7-H12/2 mice and applied to the model of Experimental Autoimmune Encephalomyelitis (EAE). Injections of B7-H1-deficient DC showed increased EAE protection as compared to wild type (WT)-DC. Injections of B7-H12/2 TNF-DC induced higher release of peptide-specific IL-10 and IL-13 after restimulation in vitro together with elevated serum cytokines IL-4 and IL-13 produced by NKT cells, and reduced IL-17 and IFN-c production in the CNS. Experiments in CD1d2/2 and Ja2812/2 mice as well as with type I and II NKT cell lines indicated that only type II NKT cells but not type I NKT cells (invariant NKT cells) could be stimulated by an endogenous CD1d-ligand on DC and were responsible for the increased serum cytokine production in the absence of B7-H1. Conclusions/Significance: Together, our data indicate that BM-DC express an endogenous CD1d ligand and B7-H1 to ihibit type II but not type I NKT cells. In the absence of B7-H1 on these DC their tolerogenic potential to stimulate tolerogenic CD4+ and NKT cell responses is enhanced

Biochemical applications of surface-enhanced infrared absorption spectroscopy

An overview is presented on the application of surface-enhanced infrared absorption (SEIRA) spectroscopy to biochemical problems. Use of SEIRA results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude and has the potential to enable new studies, from fundamental aspects to applied sciences. This report surveys studies of DNA and nucleic acid adsorption to gold surfaces, development of immunoassays, electron transfer between metal electrodes and proteins, and protein–protein interactions. Because signal enhancement in SEIRA uses surface properties of the nano-structured metal, the biomaterial must be tethered to the metal without hampering its functionality. Because many biochemical reactions proceed vectorially, their functionality depends on proper orientation of the biomaterial. Thus, surface-modification techniques are addressed that enable control of the proper orientation of proteins on the metal surface. [Figure: see text

The Society for Immunotherapy of Cancer statement on best practices for multiplex immunohistochemistry (IHC) and immunofluorescence (IF) staining and validation.

OBJECTIVES: The interaction between the immune system and tumor cells is an important feature for the prognosis and treatment of cancer. Multiplex immunohistochemistry (mIHC) and multiplex immunofluorescence (mIF) analyses are emerging technologies that can be used to help quantify immune cell subsets, their functional state, and their spatial arrangement within the tumor microenvironment. METHODS: The Society for Immunotherapy of Cancer (SITC) convened a task force of pathologists and laboratory leaders from academic centers as well as experts from pharmaceutical and diagnostic companies to develop best practice guidelines for the optimization and validation of mIHC/mIF assays across platforms. RESULTS: Representative outputs and the advantages and disadvantages of mIHC/mIF approaches, such as multiplexed chromogenic IHC, multiplexed immunohistochemical consecutive staining on single slide, mIF (including multispectral approaches), tissue-based mass spectrometry, and digital spatial profiling are discussed. CONCLUSIONS: mIHC/mIF technologies are becoming standard tools for biomarker studies and are likely to enter routine clinical practice in the near future. Careful assay optimization and validation will help ensure outputs are robust and comparable across laboratories as well as potentially across mIHC/mIF platforms. Quantitative image analysis of mIHC/mIF output and data management considerations will be addressed in a complementary manuscript from this task force