Search

Acute lymphoblastic leukemias (ALL) positive for KMT2A/AFF1 (MLL/AF4) translocation, which constitute 60% of all infant ALL cases, have a poor prognosis even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This poor prognosis is due to one of two factors, either resistance to TNFα, which mediates a graft-versus-leukemia (GVL) response after allo-HSCT, or immune resistance due to upregulated expression of the immune escape factor S100A6. Here, we report an immune stimulatory effect against KMT2A/AFF1-positive ALL cells by treatment with the anti-allergy drug amlexanox, which we found to inhibit S100A6 expression in the presence of TNF-α. In KMT2A/AFF1-positive transgenic (Tg) mice, amlexanox enhanced tumor immunity and lowered the penetrance of leukemia development. Similarly, in a NOD/SCID mouse model of human KMT2A/AFF1-positive ALL, amlexanox broadened GVL responses and extended survival. Our findings show how amlexanox degrades the resistance of KMT2A/AFF1-positive ALL to TNFα by downregulating S100A6 expression, with immediate potential implications for improving clinical management of KMT2A/AFF1-positive ALL. Cancer Res; 77(16); 4426–33. ©2017 AACR.

The purpose of this study was to investigate which of the three readily available scaffold materials would be suitable for adipose tissue engineering when implanted with adipose-derived stem cells (ASCs) in vivo. ASCs isolated from green fluorescence protein (GFP) transgenic mice were incubated in an adipogenic medium and then seeded onto type I collagen sponge, non-woven polyglycolic acid or hyaluronic acid gel. The constructs were harvested and evaluated histologically and immunohistochemically 4 and 8 weeks after subcutaneous implantation into athymic mice. The gene expression of peroxisome-proliferator-activated receptor gamma2 (PPAR-gamma2), the adipocyte-specific transcriptional factor, was also investigated by using reverse transcription-polymerase chain reaction. Histological examination showed that more adipose-tissue-like construct was regenerated when using type I collagen sponge than when the other scaffolds were used. Moreover, immunohistostaining revealed that some of the adipocytes on the type I collagen construct expressed GFP. PPAR-gamma2 gene expression in the induced ASCs in the type I collagen sponge was observed. These findings suggest that type I collagen sponge may be the most suitable among the three readily available scaffolds for adipogenesis.

Background: MLL/AF4-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis even after allogeneic hematopoietic stem cell transplantation. Previously, we reported that this ALL shows resistance to TNF-α, which is the factor involved in the graft versus leukemia (GVL) effect or tumor immunity, by upregulation of S100A6 expression followed by interference with the p53-caspase pathway. Amlexanox, an anti-allergic drug, was reported to inhibit the translocation pathway of endogenous S100A6 in endothelial cells. Aims: This study was performed to examine the effects of Amlexanox on MLL/AF4-positive ALL. Methods: In vitro analysis, cell growth of MLL/AF4-positive ALL cell lines ( SEM and RS4;11) were analyzed with TNF-α (10 ng/mL) and Amlexanox (0, 10, and 100 µg/mL).The effect of Amlexanox on S100A6 and p53-caspase pathways were examined by Western blotting (WB) analysis. In vivo analysis MLL/AF4-positive transgenic mice model, which show CD45R/B220+leukemia by 12 months of age we established and human peripheral blood mononuclear cell (Hu-PBMC) NOD/SCID mice transplanted with SEM-Luc were examined to compare mice fed diet containing Amlexanox (0.02%) with mice fed control diet. Results: There were no significant differences between the growth of SEM or RS4;11 cells in the absence or presence of 10 µg/mL of Amlexanox in vitro under 10 ng/mL of TNF-α. However, both cells showed significant growth inhibition by 10 ng/mL of TNF-α in the presence of 100 µg/mL of Amlexanox (P = 0.0085 for SEM, P = 0.0196 for RS4;11) WB analysis showed that S100A6 was activated in the presence of 10 ng/mL TNF-α, and activated S100A6 was decreased and both acetyl-p53/p53 ratio and cleaved caspase 3/caspase 3 ratio were increased in cells treated with 100 µg/mL of Amlexanox under 10 ng/mL of TNF-α in the MLL/AF4-positive human ALL cell lines. In vivo, MLL/AF4-positive transgenic mice fed a diet containing Amlexanox (0.02%) developed significantly less volume of CD45R/B220+ leukemia at the age of 1 year in comparison with mice fed control diet (P Conclusions: Amlexanox may be a breakthrough drug for MLL/AF4-positive ALL because it inhibits the resistance of MLL/AF4-positive ALL to TNF-α by downregulating S100A6 expression followed by upregulating the p53-caspase pathway.Specifically, allogeneic hematopoietic stem cell transplantation is expected to show beneficial effects in combination with Amlexanox. Disclosures No relevant conflicts of interest to declare.

Mixed-lineage leukemia (MLL)/AF4-positive acute lymphoblastic leukemia (ALL) is a common type of leukemia in infants, which is associated with a high relapse rate and poor prognosis. IL24 selectively induces apoptosis in cancer cells and exerts immunomodulatory and antiangiogenic effects. We examined the effects of adeno-associated virus type 8 (AAV8) vector-mediated muscle-directed systemic gene therapy in MLL/AF4-positive ALL using IL24. In a series of in vitro studies, we examined the effects of AAV8-IL24–transduced C2C12 cell-conditioned medium. We also examined the effects of AAV8-IL24 in MLL/AF4 transgenic mice. The results revealed the effects of AAV8-IL24 in MLL/AF4-positive ALL both in vitro and in vivo. With regard to the mechanism of therapy using AAV8-IL24 in MLL/AF4-positive ALL, we demonstrated the antiangiogenicity and effects on the ER stress pathway and unreported pathways through inhibition of S100A6 and HOXA9, which is specific to MLL/AF4-positive ALL. Inhibition of S100A6 by IL24 was dependent on TNF-α and induced acetylation of p53 followed by activation of the caspase 8–caspase 3 apoptotic pathway. Inhibition of HOXA9 by IL24, which was independent of TNF-α, induced MEIS1 activation followed by activation of the caspase 8–caspase 3 apoptotic pathway. Thus, gene therapy using AAV8-IL24 is a promising treatment for MLL/AF4-positive ALL.

Mixed-lineage leukemia (MLL)–AFF1 (MLL–AF4)-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis, even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The resistance to graft-versus-leukemia (GVL) effects may be responsible for the poor effect of allo-HSCT on MLL–AFF1-positive ALL. Cytotoxic effector mechanisms mediated by tumor necrosis factor-alpha (TNF-α) was reported to contribute to the GVL effect. We showed that MLL–AFF1-positive ALL cell lines are resistant to TNF-α. To examine the mechanism of resistance to TNF-α of MLL–AFF1-positive leukemia, we focused on S100A6 as a possible factor. Upregulation of S100A6 expression and inhibition of the p53–caspase 8–caspase 3 pathway were observed only in MLL–AFF1-positive ALL cell lines in the presence of TNF-α. The effect of S100A6 on resistance to TNF-α by inhibition of the p53–caspase 8–caspase 3 pathway of MLL–AFF1-positive ALL cell lines were also confirmed by analysis using small interfering RNA against S100A6. This pathway was also confirmed in previously established MLL–AFF1 transgenic mice. These results suggest that MLL–AFF1-positive ALL escapes from TNF-α-mediated apoptosis by upregulation of S100A6 expression, followed by interfering with p53–caspase 8–caspase 3 pathway. These results suggest that S100A6 may be a promising therapeutic target for MLL–AFF1-positive ALL in combination with allo-HSCT.

Activated K-Ras protein accelerates human MLL/AF4-induced leukemo-lymphomogenicity in a transgenic mouse model

P230 Bcr/Abl has been associated with indolent myeloproliferative disease (MPD). We generated transgenic mice expressing P230Bcr/Abl driven by the promoter of the long terminal repeat of the murine stem cell virus of the MSCV neo P230 BCR/ABL vector. Two founder mice exhibited mild granulocytosis and marked thrombocytosis and developed MPD. The disease of one founder mouse, no. 13, progressed to extramedullary myeloblastic crisis in the liver at 12 months old. The other founder mouse, no. 22, was found to have chronic-phase MPD with large populations of megakaryocytes and granulocytes in an enlarged spleen. The transgenic progeny of no. 22 clearly exhibited MPD at 15 months old. These results showed that P230Bcr/Abl had leukemogenic properties and induced MPD. The phenotype of the MPD caused by P230Bcr/Abl was characterized by mild granulocytosis, a high platelet count, infiltration of megakaryocytes in some organs, and a longer disease latency compared with the MPD caused by P210Bcr/Abl. (Blood. 2003;102:320-323)

Abstract 61 Acute lymphoblastic leukemia (ALL) with MLL/AF4 fusion is a common infant leukemia associated with a poor prognosis. Research into the molecular mechanisms underlying ALL with MLL/AF4 has been hampered by the absence of a good animal model: there is a large discrepancy between the time to disease onset in infants and the long latency seen in previously established murine models (human derived-AF4 knock-in and inverter model). We speculated that the absence of associated K-RAS mutations could explain the long latency seen in murine models of ALL with MLL/AF4. To establish a model combining MLL/AF4 fusion and activated K-RAS, we first established MLL/AF4+ transgenic (Tg) mice through overexpression of human MLL/AF4 fusion protein. These MLL/AF4+ Tg mice exhibited splenomegaly and lung tumors at 8–12 months of age (median, 12 months), and histopathological analysis showed disruption of the spleen structure and infiltration of the liver, lung and spleen by lymphoma cells consisted of pro B-cells (CD45R/B220+CD43+). Western blot analysis showed expression of both MLL/AF4 and HoxA9 was upregulated in all of the tissues tested. Infiltration of tumor cells into the peripheral blood was observed after a long latency period in MLL/AF4+ Tg mice (median, 14 months), at which time abnormal lymphocytes accounted for 7.2±1.2% (n=5) of white blood cells. FACS analysis showed these abnormal lymphocytes to be CD45R/B220+CD43+CD19+ cells, which is the same phenotype as the lymphoma cells. To develop a more aggressive oncogenic model mouse, we generated MLL/AF4+K-RAS mutation+ Tg mice by crossing MLL/AF4+ Tg mice and K-RAS mutation (G12D)+ Tg mice. The resultant mice developed lymphoma significantly earlier (median, 6 months) than MLL/AF4+ Tg mice (median LFS: 5.5 months vs. 12 months, P=0.001). Infiltration of tumor cells into the peripheral blood was observed after a short latency period in MLL/AF4+K-RAS mutation+ Tg mice (median age, 6 months), at which time leukemia cells accounted for 3.2±0.9% of WBCs, suggesting the collaborative acceleration of leukemo-lymphomogenicity through overexpression of MLL/AF4 and mutant K-RAS. We anticipate that the MLL/AF4+K-RAS mutation+ Tg mouse model will be useful for studying the molecular mechanisms of MLL/AF4 leukemogenesis and for developing new therapies against MLL-related malignancies. Disclosures: No relevant conflicts of interest to declare.