ALBERT

Description: Acute graft-versus-host diseases (GVHD) is a major cause of morbidity and mortality in patients undergoing allogeneic bone marrow transplantation (BMT). T helper 1 (Th1)-type cytokines such as interferon-γ or tumor necrosis factor-α have been implicated in the pathogenesis of acute GVHD. TAK-603 is a new quinoline derivative, which is now in clinical trials for use as a disease-modifying antirheumatic drug. In preclinical studies, it inhibited delayed-type hypersensitivity, but not Arthus-type reaction, in mice, and selectively suppressed Th1 cytokine production. Thus, the present study was designed to investigate whether the Th1 inhibitor (TAK-603) ameliorates lethal acute GVHD in a mouse model. Administration of TAK-603 into BALB/c mice given 10 Gy total body irradiation followed by transplantation of bone marrow and spleen cells from C57BL/6 mice markedly reduced the mortality in association with minimal signs of GVHD pathology in the liver, intestine, and skin. TAK-603 reduced not only the production of Th1-type cytokines, but also the proportion of Th1 cells in CD4+ helper T cells in this GVHD mouse model. These results suggest that TAK-603 could be a potent therapeutic agent for acute lethal GVHD.

Description: Signal transducer and activator of transcription (STAT) family proteins play crucial roles in the cytokine signaling pathways which regulate survival and proliferation of normal and malignant hematopoietic cells. The STAT proteins which regulate myeloid leukemia cell survival and proliferation remain ill defined. STAT3, for example, has been reported to be constitutively activated in acute myeloid leukemia (AML) cells but its function in these cells is not clear. In order to better understand the role of STAT3 in AML biology, we studied its expression, activation, and requirement for cell growth in several AML cell lines, and in primary patient material. We first confirmed the activation of STAT3 in primary AML cells by western blotting. An analysis of 5 AML patient samples revealed elevated levels of constitutive STAT3 phosphorylation in 4 of 5 samples. In addition, two AML cell lines (MOLM-14, KG-1) displayed constitutive STAT3 activation. In order to evalute the functional significance of STAT3 overexpression in AML cells, we synthesized a siRNA that had been reported effective in silencing STAT3 expression. The siRNA were delivered to MOLM-14 cells using an amaxa nucleoporation device (amaxa, Inc. Gaithersburg, MD) (Program O-17/Solution V). In treated cells, STAT3 expression decreased 72%±1% [n=5] but in a non-dose related manner suggesting that “off-target” gene silencing, or other mechanisms unrelated to target gene silencing might have played a role squelching STAT3 expression. In contrast, nucleofection of MOLM-14 cells with an antisense oligodeoxynucleotide (AS ODN) corresponding to the antisense sequence of the siRNA duplex, decreased STAT3 expression 96%±1% [n=5] compared to control treated cells. Importantly, inhibition was dose dependent and sequence specific. Cell proliferation was also inhibited in the AS ODN treated cells (82%±7% at 24 hour, 92%±2% at 48 hour, 91%±4% at 72 hour [n=3]) in comparison to control treated cells. To determine if these results were unique to a specific cell line, we nucleofected AS ODN into KG-1 cells (amaxa, Program T-27/Solution V). Again, the AS ODN decreased STAT3 expression 90% compared to control treated cells and inhibited cell proliferation in a manner similar to that obtained for MOLM-14 cells (72% at 24 hour, 78% at 48 hour, 79% at 72 hour) in comparison to control treated cells. To determine if STAT3 is necessary for survival of primary AML cells, cells from 5 patients (four frozen leukapheresis samples-FAB Classification M1, M4, M4, M5, one fresh peripheral blood sample-FAB Classification M5) were nucleofected (amaxa, Program U-15/Solution V) with the STAT3 AS or control ODN and incubated on the EBM-2(Endothelial Basal Medium, CABREX®) for 72 hours. Primary AML cells treated with STAT3 AS ODN showed a ~50–80% decrease in survival compared to control ODN treated cells. These results demonstrate that STAT3 plays an important role in the survival and proliferation of acute myeloid leukemia cells. Accordingly, STAT3 appears to be a legitimate target for the treatment of AML. These results also demonstrate that an effectively delivered, appropriately targeted, AS ODN has the ability to silence its targeted gene’s expression with a specificity and an efficiency equivalent to, or in some cases better, than any highly active siRNA.