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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.

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-␣ 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-␣, 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. IntroductionRearrangements of the mixed-lineage leukemia (MLL) gene located at 11q23 are common chromosomal abnormalities associated with acute leukemia, especially infant leukemia and secondary leukemia after treatment with DNA topoisomerase II inhibitors. In addition, 11q23/MLL abnormalities are now widely recognized as important prognostic factors in acute leukemia. More than 70 chromosomal partners of 11q23 have been identified to date, at least 50 of which have been cloned and characterized at the molecular level. 1 The prognosis of leukemia patients with MLL rearrangement varies widely depending on the partner gene, leukemia cell lineage, age of the patient, and treatment administered. 2 The most prevalent MLL rearrangement in acute lymphoblastic leukemia (ALL) generates the MLL/AF4 fusion gene because of a t(4;11)(q21;q23) chromosomal translocation. Despite recent improvements in the overall treatment outcome for ALL patients, including allogeneic HSCT (allo-HSCT), MLL/AF4-positive ALL is still associated with a poor prognosis. 2 The following are 3 factors that may be related to the poor prognosis of MLL/AF4-positive ALL. First, MLL/AF4-positive ALL cells strongly recruit new blood vessels. 3 Second, MLL/AF4-positive ALL escapes from TNF-␣-mediated apoptosis by upregulating the expression of S100A6, which is a 10.5-kDa Ca 2ϩ -binding protein belonging to the S100 protein family that has been reported to interact with and alter the conformation of p53. [4][5][6][7][8][9][10][11] Up-regulation of S100A6 expression in MLL/AF4-positive ALL inhibits p53 acetylation followed by inhibition of upregulation of the caspase 8-caspase 3 apoptotic pathway in the ...

Acute lymphoblastic leukemias (ALL) positive for () 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 -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-positive transgenic (Tg) mice, amlexanox enhanced tumor immunity and lowered the penetrance of leukemia development. Similarly, in a NOD/SCID mouse model of human -positive ALL, amlexanox broadened GVL responses and extended survival. Our findings show how amlexanox degrades the resistance of-positive ALL to TNFα by downregulating S100A6 expression, with immediate potential implications for improving clinical management of -positive ALL..