Design and testing of a humanized porcine donor for xenotransplantation.

Recent human decedent model studies1,2 and compassionate xenograft use3 have explored the promise of porcine organs for human transplantation. To proceed to human studies, a clinically ready porcine donor must be engineered and its xenograft successfully tested in nonhuman primates. Here we describe the design, creation and long-term life-supporting function of kidney grafts from a genetically engineered porcine donor transplanted into a cynomolgus monkey model. The porcine donor was engineered to carry 69 genomic edits, eliminating glycan antigens, overexpressing human transgenes and inactivating porcine endogenous retroviruses. In vitro functional analyses showed that the edited kidney endothelial cells modulated inflammation to an extent that was indistinguishable from that of human endothelial cells, suggesting that these edited cells acquired a high level of human immune compatibility. When transplanted into cynomolgus monkeys, the kidneys with three glycan antigen knockouts alone experienced poor graft survival, whereas those with glycan antigen knockouts and human transgene expression demonstrated significantly longer survival time, suggesting the benefit of human transgene expression in vivo. These results show that preclinical studies of renal xenotransplantation could be successfully conducted in nonhuman primates and bring us closer to clinical trials of genetically engineered porcine renal grafts.

Extensive germline genome engineering in pigs.

The clinical applicability of porcine xenotransplantation-a long-investigated alternative to the scarce availability of human organs for patients with organ failure-is limited by molecular incompatibilities between the immune systems of pigs and humans as well as by the risk of transmitting porcine endogenous retroviruses (PERVs). We recently showed the production of pigs with genomically inactivated PERVs. Here, using a combination of CRISPR-Cas9 and transposon technologies, we show that pigs with all PERVs inactivated can also be genetically engineered to eliminate three xenoantigens and to express nine human transgenes that enhance the pigs' immunological compatibility and blood-coagulation compatibility with humans. The engineered pigs exhibit normal physiology, fertility and germline transmission of the 13 genes and 42 alleles edited. Using in vitro assays, we show that cells from the engineered pigs are resistant to human humoral rejection, cell-mediated damage and pathogenesis associated with dysregulated coagulation. The extensive genome engineering of pigs for greater compatibility with the human immune system may eventually enable safe and effective porcine xenotransplantation.

Scavenging of Labile Heme by Hemopexin Is a Key Checkpoint in Cancer Growth and Metastases.

Hemopexin (Hx) is a scavenger of labile heme. Herein, we present data defining the role of tumor stroma-expressed Hx in suppressing cancer progression. Labile heme and Hx levels are inversely correlated in the plasma of patients with prostate cancer (PCa). Further, low expression of Hx in PCa biopsies characterizes poorly differentiated tumors and correlates with earlier time to relapse. Significantly, heme promotes tumor growth and metastases in an orthotopic murine model of PCa, with the most aggressive phenotype detected in mice lacking Hx. Mechanistically, labile heme accumulates in the nucleus and modulates specific gene expression via interacting with guanine quadruplex (G4) DNA structures to promote PCa growth. We identify c-MYC as a heme:G4-regulated gene and a major player in heme-driven cancer progression. Collectively, these results reveal that sequestration of labile heme by Hx may block heme-driven tumor growth and metastases, suggesting a potential strategy to prevent and/or arrest cancer dissemination.

A Role of the Heme Degradation Pathway in Shaping Prostate Inflammatory Responses and Lipid Metabolism.

The molecular mechanisms of prostate inflammation are unclear. We hypothesized that heme oxygenase 1 (HMOX1; HO-1), an enzyme responsible for degradation of heme to carbon monoxide, bilirubin, and iron, is an important regulator of inflammation and epithelial responses in the prostate. Injection of non-uropathogenic Escherichia coli (MG1655 strain) or phosphate-buffered saline into the urethra of mice led to increased numbers of CD45+ leukocytes and mitotic markers (phosphorylated histone H3 and phosphorylated ERK1/2) in the prostate glands. Leukocyte infiltration was elevated in the prostates harvested from mice lacking HO-1 in myeloid compartment. Conversely, exogenous carbon monoxide (250 ppm) increased IL-1ß levels and suppressed cell proliferation in the prostates. Carbon monoxide did not affect the number of infiltrating CD45+ cells in the prostates of E. coli- or phosphate-buffered saline-treated mice. Interestingly, immunomodulatory effects of HO-1 and/or carbon monoxide correlated with early induction of the long-chain acyl-CoA synthetase 1 (ACSL1). ACSL1 levels were elevated in response to E. coli treatment, and macrophage-expressed ACSL1 was in part required for controlling of IL-1ß expression and prostate cancer cell colony growth in soft agar. These results suggest that HO-1 and/or carbon monoxide might play a distinctive role in modulating prostate inflammation, cell proliferation, and IL-1ß levels in part via an ACSL1-mediated pathway.

Deletion of Biliverdin Reductase A in Myeloid Cells Promotes Chemokine Expression and Chemotaxis in Part via a Complement C5a--C5aR1 Pathway.

Biliverdin reductase (BVR)-A is a pleotropic enzyme converting biliverdin to bilirubin and a signaling molecule that has cytoprotective and immunomodulatory effects. We recently showed that biliverdin inhibits the expression of complement activation fragment 5a receptor one (C5aR1) in RAW 264.7 macrophages. In this study, we investigated the role of BVR-A in determining macrophage inflammatory phenotype and function via regulation of C5aR1. We assessed expression of C5aR1, M1-like macrophage markers, including chemokines (RANTES, IP-10), as well as chemotaxis in response to LPS and C5a in bone marrow-derived macrophages from BVR fl/fl and LysM-Cre:BVR fl / fl mice (conditional deletion of BVR-A in myeloid cells). In response to LPS, macrophages isolated from LysM-Cre:BVR fl/fl showed significantly elevated levels of C5aR1 as well as chemokines (RANTES, IP10) but not proinflammatory markers, such as iNOS and TNF. An increase in C5aR1 expression was also observed in peritoneal macrophages and several tissues from LysM-Cre:BVR fl/fl mice in a model of endotoxemia. In addition, knockdown of BVR-A resulted in enhanced macrophage chemotaxis toward C5a. Part of the effects of BVR-A deletion on chemotaxis and RANTES expression were blocked in the presence of a C5aR1 neutralizing Ab, confirming the role of C5a-C5aR1 signaling in mediating the effects of BVR. In summary, BVR-A plays an important role in regulating macrophage chemotaxis in response to C5a via modulation of C5aR1 expression. In addition, macrophages lacking BVR-A are characterized by the expression of M1 polarization-associated chemokines, the levels of which depend in part on C5aR1 signaling.

Deletion of Lactate Dehydrogenase-A in Myeloid Cells Triggers Antitumor Immunity.

Immunometabolism is emerging as a critical determinant of cancer pathophysiology. In this study, we explored the contributions of macrophage-expressed lactate dehydrogenase-A (LDH-A) to tumor formation in a K-Ras murine model of lung carcinoma. Myeloid-specific deletion of LDH-A promoted accumulation of macrophages with a CD86high and MCP-1high M1-like phenotype that suppressed tumor growth. This phenotypic effect was accompanied by reduced VEGF expression and angiogenesis, diminished numbers of PD-L1+ cancer cells, increased numbers of CD3+ T cells, and activation status of CD8+ T cells. Furthermore, it was associated with more pronounced antitumor T-cell immunity via induction of IL17 and IFNγ-producing CD8+ T (Tc17 and Tc1) cells, likely via suppression of lactate-driven PD-L1 expression. Our results suggest that expressions of LDH-A and lactate by macrophage in the tumor microenvironment are major drivers of T-cell immunosuppression, strongly supporting the concept of targeting stromal LDH-A as an effective strategy to blunt tumoral immune escape. Cancer Res; 77(13); 3632-43. ©2017 AACR.

Application of Induced Pluripotent Stem Cell Technology to the Study of Hematological Diseases.

The burst of reprogramming technology in recent years has revolutionized the field of stem cell biology, offering new opportunities for personalized, regenerative therapies. The direct reprogramming of somatic cells to induced pluripotent stem cells (iPSCs) has provided an invaluable tool to study and model a wide range of human diseases. Here, we review the transforming potential of such a strategy in research and in therapies applicable to the hematology field.

Novel synthetic chalcones induce apoptosis in the A549 non-small cell lung cancer cells harboring a KRAS mutation.

A series of novel chalcones were synthesized by the Claisen-Schmidt condensation reaction of tetralones and 5-/6-indolecarboxaldehydes. Treatment of human lung cancer cell line harboring KRAS mutation (A549) with the chalcones induced dose-dependent apoptosis. Cell cycle analyses and Western blotting suggested the critical role of the chalcones in interrupting G2/M transition of cell cycle. SAR study demonstrated that substituent on the indole N atom significantly affects the anticancer activity of the chalcones, with methyl and ethyl providing the more active compounds (EC50: 110-200nM), Compound 1g was found to be >4-fold more active in the A549 cells (EC50: 110nM) than in prostate (PC3) or pancreatic cancer (CLR2119, PAN02) cells. Furthermore, compound 1l selectively induced apoptosis of lung cancer cells A549 (EC50: 0.55µM) but did not show measurable toxicity in the normal lung bronchial epithelial cells (hBEC) at doses as high as 10µM, indicating specificity towards cancer cells.

Alterations of tumor microenvironment by carbon monoxide impedes lung cancer growth.

We hypothesized that tumor-associated macrophages (TAMs) are controlled by the diffusible gas carbon monoxide (CO). We demonstrate that induction of apoptosis in lung tumors treated with low doses of CO is associated with increased CD86 expression and activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (Erk) 1/2 pathway in tumor microenvironment. Presence of CD86-positive cells was required for the anti-tumoral effects of CO in established A549 xenografts. We show that the effects of CO on tumor stroma and reprogramming of macrophages towards the anti-tumoral phenotype is mediated by reactive oxygen species (ROS)-dependent activation of MAPK/Erk1/2-c-myc pathway as well as Notch 1-dependent negative feedback on the metabolic enzyme heme oxygenase-1 (HO-1). We find a similar negative correlation between HO-1 and active MAPK-Erk1/2 levels in human lung cancer specimens.In summary, we describe novel non-cell autonomous mechanisms by which the diffusible gas CO dictates changes in the tumor microenvironment through the modulation of macrophages.

Novel indolyl-chalcones target stathmin to induce cancer cell death.

Efficacy of current therapies for advanced and metastatic cancers remains a challenge in clinical practice. We investigated the anti-cancer potency of 3 novel indoly-chalcones (CITs). Our results indicated the lead molecule CIT-026 (Formula = C20H16FNO) induced cell death in prostate and lung cancer cell lines at sub-micromolar concentration. CITs (CIT-026, CIT-214, CIT-223) lead to microtubule destabilization, cell death and low cell proliferation, which in part was dependent on stathmin (STMN1) expression. Knockdown of STMN1 with siRNA against STMN1 in part restored viability of cancer cells in response to CITs. Further, CIT-026 and CIT-223 blocked cancer cell invasion through matrigel-coated chambers. Mechanistically, CITs inhibited phosphorylation of STMN1 leading to STMN1 accumulation and mitotic catastrophe. In summary, we have synthetized novel anti-cancer CIT molecules and defined their mechanism of action in vitro.

Heme oxygenase-1 in macrophages controls prostate cancer progression.

Innate immune cells strongly influence cancer growth and progression via multiple mechanisms including regulation of epithelial to mesenchymal transition (EMT). In this study, we investigated whether expression of the metabolic gene, heme oxygenase-1 (HO-1) in tumor microenvironment imparts significant effects on prostate cancer progression.We showed that HO-1 is expressed in MARCO-positive macrophages in prostate cancer (PCa) xenografts and human prostate cancers. We demonstrated that macrophage specific (LyzM-Cre) conditional deletion of HO-1 suppressed growth of PC3 xenografts in vivo and delayed progression of prostate intraepithelial neoplasia (PIN) in TRAMP mice. However, initiation and progression of cancer xenografts in the presence of macrophages lacking HO-1 resulted in loss of E-cadherin, a known marker of poor prognosis as well as EMT. Application of CO, a product of HO-1 catalysis, increased levels of E-cadherin in the adherens junctions between cancer cells. We further showed that HO-1-driven expression of E-cadherin in cancer cells cultured in the presence of macrophages is dependent on mitochondrial activity of cancer cells.In summary, these data suggest that HO-1-derived CO from tumor-associated macrophages influences, in part, E-cadherin expression and thus tumor initiation and progression.

Carbon monoxide induces chromatin remodelling to facilitate endothelial cell migration.

Vascular injury to vessel endothelial cells (EC), caused by either mechanical damage or chronic inflammation, is still awaiting effective therapies. In the present study we hypothesised that carbon monoxide (CO) acts on the nuclear receptor Rev-erbα to induce chromatin modification and endothelial cell migration. We demonstrate that administration of low, safe doses of exogenous CO enhances endothelial cell (EC) migration, which occurs in part through chromatin remodelling and histone H3 acetylation. Further, we show that the effects of CO are dependent on inhibition of phosphorylation of glycogen synthase kinase-3 ß (GSK3ß), activation of haem synthesis, and increased expression of Rev-erbα. Rev-erbα is a haem-containing transcription factor which in response to CO binds to target DNA, recruits the Histone Deacetylase/nuclear Receptor Corepressor (HDAC/N-CoR) complex, and regulates transcription of genes responsible for endothelial cell migration and angiogenesis. Decreased levels of Rev-erbα in chimeric mice after bone marrow transplant from Rev-erbα following bone marrow transplantation from rev-erb+/- mice resulted in loss of protective effects of CO against neointima formation after wire injury. Collectively, CO modifies chromatin structure through enhanced acetylation of histone H3 via a GSK3ß-Rev-erbα-mediated pathway to increase EC migration. We propose that CO enhances vessel repair following injury in part by regulating EPC/EC motility via Rev-erbα. Thus, inhaled CO may be beneficial in the treatment of vascular syndromes associated with dysregulated thrombosis, wound healing, and angiogenesis.