Rationale for a Combination Therapy Consisting of MCL1- and MEK-Inhibitors in Acute Myeloid Leukemia.

Amplification and overexpression of the myeloid cell leukemia differentiation protein MCL1 and the murine double minute protein MDM2 have been reported in various human tumors as well as hematological malignancies including acute myeloid leukemia (AML). While MCL1 is an anti-apoptotic member of the BCL-2 family proteins, MDM2 is an important cellular inhibitor of the p53 tumor suppressor. The key oncogene in AML is the FLT3 growth factor receptor gene. FLT3 signaling pathways including the MAPK cascade (RAS-RAF-MEK-ERK) are highly active in AML cells, leading to induced protein translation and cell proliferation as well as reduced apoptosis. Consequently, combined administration of MCL1-, MDM2-, and MEK-inhibitors may present a promising anti-leukemic treatment strategy. Here, we assessed the MCL1-antagonist S63845, the MDM2-inhibitor HDM201, and the MEK1/2-inhibitor trametinib as single agents and in combination in a variety of AML cell lines and mononuclear cells isolated from patients with hematological malignancies centered on myeloid leukemia, some lymphatic leukemia, as well as some lymphomas, for their ability to induce apoptosis and cell death. We observed a considerably varying anti-leukemic efficacy of the MCL1-inhibitor S63845 and the MEK1/2-inhibitor trametinib. Hematological cells with susceptibility to the single compounds as well as to the combined treatment were defined by elevated MCL1- and MEK-protein levels, independent of the mutational status of FLT3 and TP53. Our data indicate that hematological cells with elevated MCL1- and MEK-protein levels are most sensitive to the combined treatment with S63845 and trametinib. MCL1- and MEK1/2-protein expression may be valid biomarkers for treatment response to S63845 and trametinib, respectively.

Protection From Varicella Zoster in Solid Organ Transplant Recipients Carrying Killer Cell Immunoglobulin-Like Receptor B Haplotypes.

BACKGROUND: Natural killer cell function is regulated by inhibitory and activating killer cell immunoglobulin-like receptors (KIR). Previous studies have documented associations of KIR genotype with the risk of cytomegalovirus (CMV) replication after solid organ transplantation. METHODS: In this study of 649 solid organ transplant recipients, followed prospectively for infectious disease events within the Swiss Transplant Cohort Study, we were interested to see if KIR genotype associated with virus infections other than CMV. RESULT: We found that KIR B haplotypes (which have previously been linked to protection from CMV replication) were associated with protection from varicella zoster virus infection (hazard ratio, 0.43; 95% confidence interval, 0.21-0.91; P = 0.03). No significant associations were detected regarding the risk of herpes simplex, Epstein-Barr virus or BK polyomavirus infections. CONCLUSIONS: In conclusion, these data provide evidence that the relative protection of KIR haplotype B from viral replication after solid organ transplantation may extend beyond CMV to other herpes viruses, such as varicella zoster virus and possibly Epstein-Barr virus.

Modulation of the natural killer cell KIR repertoire by cytomegalovirus infection.

Patients carrying activating killer cell immunoglobulin-like receptor (KIR) genes are significantly protected from CMV-associated complications after solid organ or hematopoietic stem cell transplantation. Whether previous infection with CMV affects NK-cell function in healthy donors is unknown. We studied the KIR repertoire and alterations of KIR expression after in vitro exposure to CMV in 54 healthy donors. The expression of neither activating nor inhibitory KIRs was different at baseline between 23 seropositive and 31 seronegative donors. However, after co-culture of NK cells with CMV-infected fibroblast cells, expression of the inhibitory receptors KIR2DL1 and KIR2DL3 and the activating receptor KIR3DS1 significantly increased in CMV-seropositive donors. In CMV-seronegative donors, changes were subtle and restricted to the subset of NK cells expressing NK-cell group antigen 2C (NKG2C). Expansion of inhibitory KIRs occurred exclusively in donors carrying the cognate HLA class I ligands, whereas the presence of the putative ligand HLA-Bw4 was not necessary for the expansion of KIR3DS1-expressing NK cells. Our data show that previous infection with CMV does not alter the resting NK-cell receptor repertoire, but appears to modify how NK cells respond to re-exposure to CMV in vitro.

Quantity of HLA-C surface expression and licensing of KIR2DL+ natural killer cells.

Natural killer (NK) cells require interaction of inhibitory surface receptors with human leukocyte antigen (HLA) ligands during development to acquire functional competence in a process termed "licensing." The quantity of HLA required for this process is unknown. Two polymorphisms affecting HLA-C surface expression (rs9264942 and rs67384697) have recently been identified, and shown to influence progression of HIV infection. We typed a cohort of healthy donors for the two HLA-C-related polymorphisms, KIR2DL1 and KIR2DL3, and their respective HLA-C ligands and analyzed how HLA ligands influenced licensing status of killer cell immunoglobulin-like receptor (KIR)+ NK cells in terms of degranulation and cytokine production in response to HLA-deficient target cells. The presence of respective HLA class I ligands increased the function of KIR2DL1+ and KIR2DL3+ NK cells in a dose-dependent manner. In contrast, neither of the HLA-C-related polymorphisms nor the quantity of cell surface HLA-C had any significant effect on NK cell function. Interestingly, HLA-Cw7-an HLA-C allele with low surface expression-licensed KIR2DL3+ NK cells more strongly than any other KIR2DL3 ligand. The quantity of cell surface HLA-C does not appear to influence licensing of NK cells, and the HLA-C-related polymorphisms presumably influence HIV progression through factors unrelated to NK cell education.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury.

UNLABELLED: The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Vascular remodeling and antitumoral effects of mTOR inhibition in a rat model of hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is amenable to only few treatments. Inhibitors of the kinase mTOR are a new class of immunosuppressors already in use after liver transplantation. Their antiproliferative and antiangiogenic properties suggest that these drugs could be considered to treat HCC. We investigated the antitumoral effects of mTOR inhibition in a HCC model. METHODS: Hepatoma cells were implanted into livers of syngeneic rats. Animals were treated with the mTOR inhibitor sirolimus for 4 weeks. Tumor growth was monitored by MR imaging. Antiangiogenic effects were assessed in vivo by microvessel density and corrosion casts and in vitro by cell proliferation, tube formation and aortic ring assays. RESULTS: Treated rats had significantly longer survival and developed smaller tumors, fewer extrahepatic metastases and less ascites than controls. Sirolimus decreased intratumoral microvessel density resulting in extensive necrosis. Endothelial cell proliferation was inhibited at lower drug concentrations than hepatoma cells. Tube formation and vascular sprouting of aortic rings were significantly impaired by mTOR inhibition. Casts revealed that in tumors treated with sirolimus vascular sprouting was absent, whereas intussusception was observed. CONCLUSIONS: mTOR inhibition significantly reduces HCC growth and improves survival primarily via antiangiogenic effects. Inhibitors of mTOR may have a role in HCC treatment.