No abstract

We hypothesized that changes in the mitochondrial DNA (mtDNA) would significantly influence whole body metabolism, adiposity and gene expression in response to diet. Because it is not feasible to directly test these predictions in humans we used Mitochondrial-Nuclear eXchange mice, which have reciprocally exchanged nuclear and mitochondrial genomes between different Mus musculus strains. Results demonstrate that nuclear-mitochondrial genetic background combination significantly alters metabolic efficiency and body composition. Comparative RNA sequencing analysis in adipose tissues also showed a clear influence of the mtDNA on regulating nuclear gene expression on the same nuclear background (up to a 10-fold change in the number of differentially expressed genes), revealing that neither Mendelian nor mitochondrial genetics unilaterally control gene expression. Additional analyses indicate that nuclear-mitochondrial genome combination modulates gene expression in a manner heretofore not described. These findings provide a new framework for understanding complex genetic disease susceptibility.

FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anti-cancer agent in multiple animal models. Our published work indicates that the anti-cancer effects of FTY720 do not depend on actions at S1P receptors, but instead stem from FTY720’s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720’s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.

FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has anti-neoplastic actions. Our published work suggests that at least some of FTY720’s anti-cancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720’s S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective anti-tumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for anti-leukemic activity.

The frequency of poor outcomes in relapsed leukemia patients underscores the need for novel therapeutic approaches. The FDA-approved immunosuppressant FTY720 limits leukemia progression by activating protein phosphatase 2A and restricting nutrient access. Unfortunately, FTY720 cannot be re-purposed for use in cancer patients due to on-target toxicity associated with S1P receptor activation at the elevated, anti-neoplastic dose. Here we show that the constrained azacyclic FTY720 analog SH-RF-177 lacks S1P receptor activity but maintains anti-leukemic activity in vitro and in vivo. SH-RF-177 was not only more potent than FTY720, but killed via a distinct mechanism. Phosphorylation is dispensable for FTY720’s anti-leukemic actions. However, chemical biology and genetic approaches demonstrated that the sphingosine kinase 2- (SPHK2) mediated phosphorylation of SH-RF-177 led to engagement of a pro-apoptotic target and increased potency. The cytotoxicity of membrane-permeant FTY720 phosphonate esters suggests that the enhanced potency of SH-RF-177 stems from its more efficient phosphorylation. The tight inverse correlation between SH-RF-177 IC50 and SPHK2 mRNA expression suggests a useful biomarker for SH-RF-177 sensitivity. In summary, these studies indicate that FTY720 analogs that are efficiently phosphorylated but fail to activate S1P receptors may be superior anti-leukemic agents compared to compounds that avoid cardiotoxicity by eliminating phosphorylation.

Nutrient stress that produces quiescence and catabolism in normal cells is lethal to cancer cells because oncogenic mutations constitutively drive anabolism. One driver of biosynthesis in cancer cells is the mTORC1 signaling complex. Activating mTORC1 by deleting its negative regulator TSC2 leads to hypersensitivity to glucose deprivation. We have previously shown that ceramide kills cells in part by triggering nutrient transporter loss and restricting access to extracellular amino acids and glucose suggesting that TSC2-deficient cells would be hypersensitive to ceramide. However, murine embryonic fibroblasts (MEFs) lacking TSC2 were highly resistant to ceramide-induced death. Consistent with the observation that ceramide limits access to both amino acids and glucose, TSC2−/− MEFs also had a survival advantage when extracellular amino acids and glucose were both reduced. As TSC2−/− MEFs were resistant to nutrient stress despite sustained mTORC1 activity, we assessed whether mTORC1 signaling might be beneficial under these conditions. In low amino acid and glucose medium and following ceramide-induced nutrient transporter loss, elevated mTORC1 activity significantly enhanced the adaptive up-regulation of new transporter proteins for amino acids and glucose. Strikingly, the introduction of oncogenic Ras abrogated the survival advantage of TSC2−/− MEFs upon ceramide treatment most likely by increasing nutrient demand. These results suggest that, in the absence of oncogene-driven biosynthetic demand, mTORC1 dependent translation facilitates the adaptive cellular response to nutrient stress.

MicroRNAs (miRNAs, miRs) regulate cell fate decisions by post-transcriptionally tuning networks of mRNA targets. We used miRNA-directed pathway discovery to reveal a regulatory circuit that influences Ig class switch recombination (CSR). We developed a system to deplete mature, activated B cells of miRNAs, and performed a rescue screen that identified the miR-221/222 family as a positive regulator of CSR. Endogenous miR-221/222 regulated B cell CSR to IgE and IgG1 in vitro, and miR-221/222–deficient mice exhibited defective IgE production in allergic airway challenge and polyclonal B cell activation models in vivo. We combined comparative Ago2-HITS-CLIP and gene expression analyses to identify mRNAs bound and regulated by miR-221/222 in primary B cells. Interrogation of these putative direct targets uncovered functionally relevant downstream genes. Genetic depletion or pharmacological inhibition of Foxp1 and Arid1a confirmed their roles as key modulators of CSR to IgE and IgG1.

Sickle cell nephropathy (SCN) is a major complication of sickle cell disease (SCD). However, it is not fully understood what role immune cells play in SCN. Recent studies have implicated IL‐17A+CD4+(TH17) cells in mediating kidney disease outside of SCD. Furthermore, levels of circulating TH17 cells are elevated in SCD patients. We have previously reported that humanized sickle cell (HbSS) mice show progressive age‐dependent nephropathy with proteinuria, glomerulosclerosis, loss of GFR, and elevated CD3+cells compared to control (HbAA) mice. However, a role for renal TH17 cells in SCN has not yet been defined. We hypothesized that HbSS mice have elevated renal TH17 cells compared to HbAA mice. Thus, we phenotyped renal and splenic lymphocytes from 16–24 week‐old male HbAA and HbSS mice by flow cytometry. HbSS mice had increased renal TH17 cells compared to HbAA mice (18.3 ± 2.0% vs. 0.2 ± 0.1%, respectively; p<0.001) but no difference in renal CD4+cells. No differences were observed in splenic TH17 cells between HbSS and HbAA mice, which correlated with similar frequencies of splenic CD4+cells. Interestingly, HbSS mice had a significant increase in renal CXCR3+CD4+(TH1) cells compared to HbAA mice (48.8 ± 2.3% vs. 10.9 ± 3.3%, respectively; p<0.001) but no difference in splenic TH1 cells. Next, we measured changes in circulating proinflammatory mediators by Luminex multiplex technology. HbSS mice had elevated levels of soluble intercellular adhesion molecule 1 (sICAM‐1: 2937 ± 240 ng/mL vs. 2396 ± 57 ng/mL, respectively; p=0.04 ) and plasminogen activator inhibitor 1 (PAI‐1: 546.9 ± 120 ng/mL vs. 69.6 ± 4.5 ng/mL; p=0.001 ) compared to HbAA mice. We conducted a preliminary flow cytometric analysis of activated renal and splenic CD4+cells from aged (8–13 month‐old) mice and observed decreased splenic CD4+cells in aged HbSS mice compared to aged HbAA mice (5.1% vs. 32.5%, respectively; n = 2) but no differences in renal CD4+cells. Activated CD44hiCD4+cells from aged HbSS mice were increased in both the spleen (68.8% vs. 30.8%; n = 2) and the kidney (22.2% vs. 14.0%, respectively; n = 2) compared to aged HbAA mice. We propose that these findings and initial observations suggest an important role for renal T cells, especially TH17 and TH1 cells, in promoting and exacerbating kidney injury and dysfunction in SCD.Support or Funding InformationSupport provided by NIH T32 DK116672 to PAM, F30 DK107194 to BMF, NIH P01 HL136267 to CDM, JSP, DMP, NIH T32 AR069516 to RJM, NIH U01 HL117684 to JSP, DMP, and ASN Ben J. Lipps Research Fellowship to MK.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.