642 Decreased host PKC-delta is associated with the T cell-inflamed tumor microenvironment and improves anti-tumor immunity

BackgroundFavorable clinical responses to immunotherapy have been correlated with a T cell-inflamed tumor microenvironment. The degree of spontaneous immune infiltration in tumors varies widely between individual patients. We hypothesized that germline polymorphisms in immune regulatory genes may affect the host immune response to solid tumors, similar to their influence on autoimmune susceptibility.MethodsMelanoma TCGA RNAseq and germline SNP data were utilized to identify germline polymorphisms associated with the magnitude of an immune gene signature score. The top GWAS hit associated with increased immune gene expression in melanoma was SNP rs1483185 (p = 8.812e-08, Bonferroni corrected <0.05), within the PKC-delta gene. Using a lymphoblastic cell line GTEX database, this SNP was associated with lower expression PKC-delta, implying a loss of function phenotype. Germline mutations in PKC-delta had previously been associated with familial lupus. To study the role of PKC-delta in anti-tumor immunity, knockout hematopoietic cells and conditional knockout mice were utilized, and implanted tumors were monitored along with detailed immune response analysis.ResultsB16.SIY tumors grew more slowly in chimeras reconstituted with PKC-delta-/- bone marrow compared to WT bone marrow, and this effect was dependent on CD8-beta+ cells. T cell priming in the tumor-draining lymph node was comparable in WT and KO hosts. However, tumors in PKC-delta-/- bone marrow chimeras had increased numbers of CD8+ T cells in the tumor at endpoint, and also responded better to anti-PD-L1 therapy. Single cell RNAseq of the tumor microenvironment revealed that PKC-delta loss primarily altered gene expression in myeloid cell subsets, leading to to increased expression of M1 associated genes and decreased expression of M2 associated genes in PKC-delta-/- chimeras. To follow up further, a conditional PKC-delta KO mouse was developed and crossed to the hematopoietic Vav1-iCre and also to LysM-Cre transgenic mice. In both instances, immune-mediated tumor control was improved, demonstrating that loss of PKC-delta in the myeloid compartment is sufficient to recapitulate the phenotype.ConclusionsOur results demonstrate that germline variants in immune regulatory genes can profoundly affect anti-tumor immunity and the efficacy of PD-1/PD-L1 blockade. In particular, the myeloid-expressed molecule PKC-delta plays an important regulatory role such that decreased expression/activity mediates improved anti-tumor immunity by altering the M1/M2 ratio. The development of pharmacologic approaches to phenocopy this loss of function phenotype may be attractive to pursue as a novel therapeutic strategy.Ethics ApprovalUniversity of Chicago IRB Protocol 15-0837

Normal spontaneous firing of cardiac pacemaker cells is regulated by basal pkc delta activation

The spontaneous beating rate of rabbit sinoatrial node cells (SANC) is regulated by local subsarcolemmal calcium releases (LCRs) from sarcoplasmic reticulum (SR). LCRs appear during diastolic depolarization (DD) and activate an inward sodium/calcium exchange current which increases DD rate and thus accelerates spontaneous SANC firing. High basal level of protein kinase A and calcium/calmodulin-dependent protein kinase II phosphorylation are required to sustain basal LCRs and normal spontaneous SANC firing. Recently we discovered that basal PKC activation is also obligatory for cardiac pacemaker function: inhibition of PKC activity by broad spectrum PKC inhibitors Bis I or calphostin C markedly suppressed SR calcium cycling and decreased or abolished spontaneous beating of freshly isolated rabbit SANC. Here we studied which PKC isoforms mediate PKC-dependent effects on cardiac pacemaker cell automaticity. The PKC superfamily consists of 3 major subgroups: conventional, novel and atypical. All PKC isoforms were detected at the RNA level (RT-qPCR) in the rabbit SA node and ventricle, and expression levels were comparable in both tissues. Expression of PKCβ, however, was markedly higher in the rabbit SA node, compared to other PKC isoenzymes in either tissue. We verified expression of conventional PKC (α, β) and novel PKC-delta at the protein level in SANC and ventricular myocytes (VM). Western blot confirmed RNA results, showing a 6-fold higher PKCβ protein abundance in SANC compared to VM. Expression of PKCα protein was similar in both cell types, while PKC-delta protein was more abundant in VM. To study whether PKCβ regulates spontaneous beating of SANC we employed selective inhibitor of conventional (α, β, gamma) PKC isoforms Go6976 (10 μmol/L), which had no effects on either LCR characteristics (confocal microscopy, calcium indicator Fluo-3AM) or spontaneous beating of freshly isolated rabbit SANC (perforated patch-clamp technique). Because selective PKC-delta inhibitors are not available, we explored effects of PKC-delta inhibition comparing effects of Go6976 (the inhibitor of conventional PKCs) and Go6983, which inhibits conventional PKCs and PKC-delta. In contrast to Go6976, Go6983 (5 μmol/L) markedly decreased the LCR size (from 7.1±0.4 to 4.5±0.3 μm) and number per each spontaneous cycle (from 1.3±0.1 to 0.8±0.1). It also markedly increased the LCR period (time from the prior AP-induced calcium transient to the subsequent LCR) which was paralleled by an increase in the spontaneous SANC cycle length. Rottlerin, another PKC-delta inhibitor, produced similar effects on LCR characteristics, and markedly and time-dependently decreased DD rate, leading to an increase in the spontaneous cycle length, and finally abrogated the spontaneous SANC firing. Thus, our data indicate that basal activity of PKC-delta, but not that of PKCβ, is essential for generation of LCRs and normal spontaneous firing of cardiac pacemaker cells. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Intramural Research Program, National Institute on Aging, National Institute of Health, USA

Pivarubicin is more effective than doxorubicin against triple-negative breast cancer in vivo

Background: Triple-negative breast cancer (TNBC) is unresponsive to anti-estrogen and anti-HER2 therapies, requiring the use of cytotoxic drug combinations of anthracyclines, taxanes, cyclophosphamide and platinum compounds. Multidrug therapies achieve pathological cure rates of only 20-40%, a consequence of drug resistance and cumulative dose limitations necessitated by the irreversible cardiotoxic effects of anthracyclines and other cytotoxic agents. Safer and more effective treatments for TNBC are required to achieve durable therapeutic responses. This study describes the mechanism of action and in vivo efficacy of pivarubicin, a structurally and functionally novel anthracycline, to determine whether pivarubicin is potentially more effective and safer than doxorubicin against human primary TNBC. Methods: Hydrolytic stability, mechanism and ability of pivarubicin to circumvent mechanisms of resistance are tested in multiple tumor lines through modulation of PKC-delta activity and assessment of drug cytotoxicity. Comparative in vivo efficacy is tested in an orthotopic NSG mouse model implanted with MDA-MB-231 human TNBC cells and treated with the maximum tolerated doses of pivarubicin and doxorubicin, followed by monitoring of tumor growth by digital caliper measurements and determination of endpoint tumor weight and volume. Endpoint cardiotoxicity is assessed histologically by identifying microvacuolization in ventricular cardiomyocytes. Results: The trimethylester moiety of pivarubicin confers hydrolytic stability relative to the closely related congener, AD 198, but retains the ability of other N -alkylbenzyladriamycin compounds to directly activate PKC-delta and trigger rapid mitochondrial-dependent apoptosis. The structure and function of pivarubicin permits circumvention of resistance conferred by overexpression of P-glycoprotein, Bcl-2, Bcl-X L and Bcr-Abl. Primary tumors treated with the multiple rounds of the maximum tolerated dose (MTD) of doxorubicin failed to inhibit tumor growth compared with vehicle-treated tumors. However, administration of a single MTD of pivarubicin produced significant inhibition of tumor growth and tumor regression relative to tumor volume prior to initiation of treatment. Histological analysis of hearts excised from drug- and vehicle-treated mice revealed that pivarubicin produced no evidence of myocardial damage at this therapeutic dose. Conclusion: These results support the development of pivarubicin as a safer and more effective replacement for doxorubicin against TNBC as well as other malignancies for which doxorubicin therapy is indicated.

Determination of the Role and Active Sites of PKC-Delta-Like from Lamprey in Innate Immunity

Protein kinase C-δ (PKC-δ) is an important protein in the immune system of higher vertebrates. Lampreys, as the most primitive vertebrates, have a uniquevariable lymphocyte receptor (VLR) immune system. PKC-δ-like is a crucial functional gene in lampreys and is highly expressed in their immune organs. In this study, lampreys were stimulated with different immunogens, and lipopolysaccharide (LPS) was found to increase the expression of PKC-δ-like. Overexpression of PKC-δ-like could also effectively activate the innate immune response. We further demonstrated that PKC-δ-like-CF, a catalytic fragment of PKC-δ-like, is responsible for activating the innate immune response, and Thr-211, which is Thr-419 of PKC-δ-like, was confirmed to be the key site affecting PKC-δ-like-CF activity. These results indicated that PKC-δ-like from lamprey may have an important role in the innate immune response.

Aldosterone rapidly activates p-PKC delta and GPR30 but suppresses p-PKC epsilon protein levels in rat kidney

Objectives. Aldosterone rapidly enhances protein kinase C (PKC) alpha and beta1 proteins in the rat kidney. The G protein-coupled receptor 30 (GPR30)-mediated PKC pathway is involved in the inhibition of the potassium channel in HEK-239 cells. GPR30 mediates rapid actions of aldosterone in vitro. There are no reports available regarding the aldosterone action on other PKC isoforms and GPR30 proteins in vivo. The aim of the present study was to examine rapid actions of aldosterone on protein levels of phosphorylated PKC (p-PKC) delta, p-PKC epsilon, and GPR30 simultaneously in the rat kidney.Methods. Male Wistar rats were intraperitoneally injected with normal saline solution or aldosterone (150 µg/kg body weight). After 30 minutes, abundance and immunoreactivity of p-PKC delta, p-PKC epsilon, and GPR30 were determined by Western blot analysis and immunohisto-chemistry, respectively.Results. Aldosterone administration significantly increased the renal protein abundance of p-PKC delta by 80% (p<0.01) and decreased p-PKC epsilon protein by 50% (p<0.05). Aldosterone injection enhanced protein immunoreactivity of p-PKC delta but suppressed p-PKC epsilon protein intensity in both kidney cortex and medulla. Protein abundance of GPR30 was elevated by aldosterone treatment (p<0.05), whereas the immunoreactivity was obviously changed in the kidney cortex and inner medulla. Aldosterone translocated p-PKC delta and GPR30 proteins to the brush border membrane of proximal convoluted tubules.Conclusions. This is the first in vivo study simultaneously demonstrating that aldosterone administration rapidly elevates protein abundance of p-PKC delta and GPR30, while p-PKC epsilon protein is suppressed in rat kidney. The stimulation of p-PKC delta protein levels by aldosterone may be involved in the activation of GPR30.