In Vitro Induction of Human Regulatory T Cells Using Conditions of Low Tryptophan Plus Kynurenines.

Thymic regulatory T cells (tTregs) and induced regulatory T cells (iTregs) suppress murine acute graft-versus-host disease (GVHD). Previously, we demonstrated that the plasmacytoid dendritic cell indoleamine 2,3-dioxygenase (IDO) fosters the in vitro development of human iTregs via tryptophan depletion and kynurenine (Kyn) metabolites. We now show that stimulation of naïve CD4+ T cells in low tryptophan (low Trp) plus Kyn supports human iTreg generation. In vitro, low Trp + Kyn iTregs and tTregs potently suppress T effector cell proliferation equivalently but are phenotypically distinct. Compared with tTregs or T effector cells, bioenergetics profiling reveals that low Trp + Kyn iTregs have increased basal glycolysis and oxidative phosphorylation and use glutaminolysis as an energy source. Low Trp + Kyn iTreg viability was reliant on interleukin (IL)-2 in vitro. Although in vivo IL-2 administration increased low Trp + Kyn iTreg persistence on adoptive transfer into immunodeficient mice given peripheral blood mononuclear cells to induce GVHD, IL-2-supported iTregs did not improve recipient survival. We conclude that low Trp + Kyn create suppressive iTregs that have high metabolic needs that will need to be addressed before clinical translation.

Cytosolic malate dehydrogenase activity helps support glycolysis in actively proliferating cells and cancer.

Increased glucose consumption is a hallmark of cancer cells. The increased consumption and subsequent metabolism of glucose during proliferation creates the need for a constant supply of NAD, a co-factor in glycolysis. Regeneration of the NAD required to support enhanced glycolysis has been attributed to the terminal glycolytic enzyme, lactate dehydrogenase (LDH). However, loss of glucose carbons to biosynthetic pathways early in glycolysis reduces the carbon supply to LDH. Thus, alternative routes for NAD regeneration must exist to support the increased glycolytic rate while allowing for the diversion of glucose to generate biomass and support proliferation. Here we demonstrate, using a variety of cancer cell lines as well as activated primary T cells, that cytosolic malate dehydrogenase 1 (MDH1) is an alternative to LDH as a supplier of NAD. Moreover, our results indicate that MDH1 generates malate with carbons derived from glutamine, thus enabling utilization of glucose carbons for glycolysis and for biomass. Amplification of MDH1 occurs at an impressive frequency in human tumors and correlates with poor prognosis. Together, our findings suggest that proliferating cells rely on both MDH1 and LDH to replenish cytosolic NAD, and that therapies designed at targeting glycolysis must consider both dehydrogenases.

Transcription factor C/EBP-ß induces tumor-suppressor phosphatase PHLPP2 through repression of the miR-17-92 cluster in differentiating AML cells.

PHLPP2, a member of the PH-domain leucine-rich repeat protein phosphatase (PHLPP) family, which targets oncogenic kinases, has been actively investigated as a tumor suppressor in solid tumors. Little is known, however, regarding its regulation in hematological malignancies. We observed that PHLPP2 protein expression, but not its mRNA, was suppressed in late differentiation stage acute myeloid leukemia (AML) subtypes. MicroRNAs (miR or miRNAs) from the miR-17-92 cluster, oncomir-1, were shown to inhibit PHLPP2 expression and these miRNAs were highly expressed in AML cells that lacked PHLPP2 protein. Studies showed that miR-17-92 cluster regulation was, surprisingly, independent of transcription factors c-MYC and E2F in these cells; instead all-trans-retinoic acid (ATRA), a drug used for terminally differentiating AML subtypes, markedly suppressed miR-17-92 expression and increased PHLPP2 protein levels and phosphatase activity. Finally, we demonstrate that the effect of ATRA on miR-17-92 expression is mediated through its target, transcription factor C/EBPß, which interacts with the intronic promoter of the miR-17-92 gene to inhibit transactivation of the cluster. These studies reveal a novel mechanism for upregulation of the phosphatase activity of PHLPP2 through C/EBPß-mediated repression of the miR-17-92 cluster in terminally differentiating myeloid cells.