Enhanced ALOX12 Gene Expression Predicts Therapeutic Susceptibility to 5-Azacytidine in Patients with Myelodysplastic Syndromes.

5-azacytidine (AZA), a representative DNA-demethylating drug, has been widely used to treat myelodysplastic syndromes (MDS). However, it remains unclear whether AZA's DNA demethylation of any specific gene is correlated with clinical responses to AZA. In this study, we investigated genes that could contribute to the development of evidence-based epigenetic therapeutics with AZA. A DNA microarray identified that AZA specifically upregulated the expression of 438 genes in AZA-sensitive MDS-L cells but not in AZA-resistant counterpart MDS-L/CDA cells. Of these 438 genes, the ALOX12 gene was hypermethylated in MDS-L cells but not in MDS-L/CDA cells. In addition, we further found that (1) the ALOX12 gene was hypermethylated in patients with MDS compared to healthy controls; (2) MDS classes with excess blasts showed a relatively lower expression of ALOX12 than other classes; (3) a lower expression of ALOX12 correlated with higher bone marrow blasts and a shorter survival in patients with MDS; and (4) an increased ALOX12 expression after AZA treatment was associated with a favorable response to AZA treatment. Taking these factors together, an enhanced expression of the ALOX12 gene may predict favorable therapeutic responses to AZA therapy in MDS.

Reprogramming of pyrimidine nucleotide metabolism supports vigorous cell proliferation of normal and malignant T cells.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is triggered by infection with human T-cell lymphotropic virus-1 (HTLV-1). Here, we describe the reprogramming of pyrimidine biosynthesis in both normal T cells and ATL cells through regulation of uridine-cytidine kinase 2 (UCK2), which supports vigorous proliferation. UCK2 catalyzes the monophosphorylation of cytidine/uridine and their analogues during pyrimidine biosynthesis and drug metabolism. We found that UCK2 was overexpressed aberrantly in HTLV-1-infected T cells but not in normal T cells. T-cell activation via T-cell receptor (TCR) signaling induced expression of UCK2 in normal T cells. Somatic alterations and epigenetic modifications in ATL cells activate TCR signaling. Therefore, we believe that expression of UCK2 in HTLV-1-infected cells is induced by dysregulated TCR signaling. Recently, we established azacitidine-resistant (AZA-R) cells showing absent expression of UCK2. AZA-R cells proliferated normally in vitro, whereas UCK2 knockdown inhibited ATL cell growth. Although uridine and cytidine accumulated in AZA-R cells, possibly because of dysfunction of pyrimidine salvage biosynthesis induced by loss of UCK2 expression, the amount of UTP and CTP was almost the same as in parental cells. Furthermore, AZA-R cells were more susceptible to an inhibitor of dihydroorotic acid dehydrogenase, which performs the rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, and more resistant to dipyridamole, an inhibitor of pyrimidine salvage biosynthesis, suggesting that AZA-R cells adapt to UCK2 loss by increasing de novo pyrimidine nucleotide biosynthesis. Taken together, the data suggest that fine-tuning pyrimidine biosynthesis supports vigorous cell proliferation of both normal T cells and ATL cells.

A Combination of Alectinib and DNA-Demethylating Agents Synergistically Inhibits Anaplastic-Lymphoma-Kinase-Positive Anaplastic Large-Cell Lymphoma Cell Proliferation.

The recent evolution of molecular targeted therapy has improved clinical outcomes in several human malignancies. The translocation of anaplastic lymphoma kinase (ALK) was originally identified in anaplastic large-cell lymphoma (ALCL) and subsequently in non-small cell lung carcinoma (NSCLC). Since ALK fusion gene products act as a driver of carcinogenesis in both ALCL and NSCLC, several ALK tyrosine kinase inhibitors (TKIs) have been developed. Crizotinib and alectinib are first- and second-generation ALK TKIs, respectively, approved for the treatment of ALK-positive ALCL (ALK+ ALCL) and ALK+ NSCLC. Although most ALK+ NSCLC patients respond to crizotinib and alectinib, they generally relapse after several years of treatment. We previously found that DNA-demethylating agents enhanced the efficacy of ABL TKIs in chronic myeloid leukemia cells. Moreover, aberrant DNA methylation has also been observed in ALCL cells. Thus, to improve the clinical outcomes of ALK+ ALCL therapy, we investigated the synergistic efficacy of the combination of alectinib and the DNA-demethylating agent azacytidine, decitabine, or OR-2100 (an orally bioavailable decitabine derivative). As expected, the combination of alectinib and DNA-demethylating agents synergistically suppressed ALK+ ALCL cell proliferation, concomitant with DNA hypomethylation and a reduction in STAT3 (a downstream target of ALK fusion proteins) phosphorylation. The combination of alectinib and OR-2100 markedly altered gene expression in ALCL cells, including that of genes implicated in apoptotic signaling, which possibly contributed to the synergistic anti-ALCL effects of this drug combination. Therefore, alectinib and OR-2100 combination therapy has the potential to improve the outcomes of patients with ALK+ ALCL.

Combination of a New Oral Demethylating Agent, OR2100, and Venetoclax for Treatment of Acute Myeloid Leukemia.

The standard treatment for elderly patients with acute myeloid leukemia (AML) is venetoclax (Ven), a BCL-2-selective inhibitor, combined with hypomethylating agents (HMA) such as azacitidine or decitabine. This regimen results in low toxicity, high response rates, and potentially durable remission; however, because of low oral bioavailability, these conventional HMAs must be administered intravenously or subcutaneously. A combination of oral HMAs and Ven would provide a therapeutic advantage over parenteral administration of drugs and improve quality of life by reducing the number of hospital visits. Previously, we showed the promising oral bioavailability and antileukemia effects of a new HMA, OR2100 (OR21). Here, we investigated the efficacy and underlying mechanism of OR21 when used in combination with Ven to treat AML. OR21/Ven showed synergistic antileukemia effects in vitro, and significantly prolonged survival without increasing toxicity in a human leukemia xenograft mice model. RNA sequencing following combination therapy revealed downregulation of VAMP7, which is involved in autophagic maintenance of mitochondrial homeostasis. Combination therapy led to accumulation of reactive oxygen species, leading to increased apoptosis. The data suggest that the combination of OR21 plus Ven is a promising candidate oral therapy for AML. Significance: The standard treatment for elderly patients with AML is Ven combined with HMAs. OR21, a new oral HMA plus Ven showed synergistic antileukemia effects in vitro and vivo, suggesting that the combination of OR2100 plus Ven is a promising candidate oral therapy for AML.

Dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth in ATL.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4+ T cells caused by human T-cell lymphotropic virus type 1 (HTLV-1)-induced T-cell transformation. After infection with HTLV-1, it takes several decades for HTLV-1 carriers to develop ATL. The prognosis of ATL remains poor despite several new agents being approved in the last few years. Recently, it has been noted that epigenetic abnormalities, both DNA methylation and trimethylation at histone H3Lys27 (H3K27me3), contribute to ATL leukemogenesis. Here, we investigated the effect of combination treatment with DNA demethylating agents (azacitidine [AZA], decitabine (DAC), and OR-2100 (OR21), which is a silylated derivative of DAC) and inhibitors of enhancer of zeste homolog 2 (EZH2) (EPZ-6438 and DS-3201b), which catalyze trimethylation of H3K27, in ATL. The combination of DAC and OR21 but not AZA with EZH inhibitors exhibited synergistic anti-ATL effects in vitro and in vivo, concomitant with DNA demethylation and reduction of H3K27me3. The combination induced gene expression reprogramming. Dual-specificity phosphatase 5 (DUSP5), an extracellular signal-regulated kinase (ERK)-specific phosphatase, was identified as a key molecule that mediated the inhibitory effect of combination treatment by inactivating the ERK signaling pathway. DUSP5 was downregulated by DNA methylation and H3K27me3 accumulation in the promoter region in HTLV-1-infected cells from patients with ATL during ATL leukemogenesis. The present results demonstrate that dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth by restoring DUSP5, and that dual targeting of aberrant DNA and histone methylation is a feasible therapeutic approach for ATL.

Adult T-cell leukemia-lymphoma acquires resistance to DNA demethylating agents through dysregulation of enzymes involved in pyrimidine metabolism.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive neoplasm derived from T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Recently, we reported that regional DNA hypermethylation in HTLV-1-infected T-cells reflects the disease status of ATL and the anti-ATL effects of DNA demethylating agents, including azacitidine (AZA), decitabine (DAC) and a new DAC prodrug, OR-2100 (OR21), which we developed. Here, to better understand the mechanisms underlying drug resistance, we generated AZA-, DAC- and OR21-resistant (AZA-R, DAC-R and OR21-R, respectively) cells from the ATL cell line TL-Om1 and the HTLV-1-infected cell line MT-2 via long-term drug exposure. The efficacy of OR21 was almost the same as that of DAC, indicating that the pharmacodynamics of OR21 were due to release of DAC from OR21. Resistant cells did not show cellular responses observed in parental cells induced by treatment with drugs, including growth suppression, depletion of DNA methyltransferase DNMT1 and DNA hypomethylation. We also found that reduced expression of deoxycytidine kinase (DCK) correlated with lower susceptibility to DAC/OR21 and that reduced expression of uridine cytidine kinase2 (UCK2) correlated with reduced susceptibility to AZA. DCK and UCK2 catalyze phosphorylation of DAC and AZA, respectively; reconstitution of expression reversed the resistant phenotypes. A large homozygous deletion in DCK and a homozygous splice donor site mutation in UCK2 were identified in DAC-R TL-Om1 and AZA-R TL-Om1, respectively. Both genomic mutations might lead to loss of protein expression. Thus, inactivation of UCK2 and DCK might be a putative cause of phenotypes that are resistant to AZA and DAC/OR21, respectively.

Silylation of Deoxynucleotide Analog Yields an Orally Available Drug with Antileukemia Effects.

DNA methyltransferase inhibitors have improved the prognosis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, because these agents are easily degraded by cytidine deaminase (CDA), they must be administered intravenously or subcutaneously. Recently, two orally bioavailable DNA methyltransferase inhibitors, CC-486 and ASTX727, were approved. In previous work, we developed 5-O-trialkylsilylated decitabines that resist degradation by CDA. However, the effects of silylation of a deoxynucleotide analog and enzymatic cleavage of silylation have not been fully elucidated. Enteric administration of OR21 in a cynomolgus monkey model led to high plasma concentrations and hypomethylation, and in a mouse model, oral administration of enteric-coated OR21 led to high plasma concentrations. The drug became biologically active after release of decitabine (DAC) from OR21 following removal of the 5'-O-trisilylate substituent. Toxicities were tolerable and lower than those of DAC. Transcriptome and methylome analysis of MDS and AML cell lines revealed that OR21 increased expression of genes associated with tumor suppression, cell differentiation, and immune system processes by altering regional promoter methylation, indicating that these pathways play pivotal roles in the action of hypomethylating agents. OR21 induced cell differentiation via upregulation of the late cell differentiation drivers CEBPE and GATA-1 Thus, silylation of a deoxynucleotide analog can confer oral bioavailability without new toxicities. Both in vivo and in vitro, OR21 exerted antileukemia effects, and had a better safety profile than DAC. Together, our findings indicate that OR21 is a promising candidate drug for phase I study as an alternative to azacitidine or decitabine.

Acute myeloid leukaemia presenting with ecthyma gangrenosum as the first manifestation: A case report.

RATIONALE: Ecthyma gangrenosum (EG) is an uncommon cutaneous infection usually associated with Pseudomonas aeruginosa bacteremia in immunocompromised patients, particularly those with underlying malignant diseases. Despite its rarity, especially in immunocompetent or nondiagnosed immunodeficiency patients, EG can present as the first manifestation of an underlying immunosuppression. PATIENT CONCERNS: A 42-year-old Japanese man was admitted to our hospital with a 3-day history of a painless red macule on his right forearm and fever. DIAGNOSES: Blood culture on admission revealed the presence of Pseudomonas aeruginosa, whereas pus culture of the skin lesion showed Pseudomonas aeruginosa and methicillin-susceptible Staphylococcus aureus positivity. INTERVENTIONS: Additional bone marrow aspirate examination and immunophenotyping were performed to confirm the diagnosis of acute promyelocytic leukaemia with PML-retinoic acid alpha receptor. OUTCOMES: The patient was successfully treated with a 14-day course of antibiotics, and no evidence of relapse was noted. The patient achieved complete remission after treatment for acute promyelocytic leukaemia. LESSONS: It should be kept in mind that EG is an important cutaneous infection that is typically associated with P aeruginosa bacteremia and the presence of underlying immunodeficiency, such as acute leukaemia.