Maternal embryonic leucine-zipper kinase (MELK), which was reported to be frequently up-regulated in various types of solid cancer, plays critical roles in formation and maintenance of cancer stem cells. However, little is known about the relevance of this kinase in hematologic malignancies. Here we report characterization of possible roles of MELK in acute myeloid leukemia (AML). MELK is expressed in AML cell lines and AML blasts with higher levels in less differentiated cells. MELK is frequently upregulated in AML with complex karyotypes and is associated with worse clinical outcome. MELK knockdown resulted in growth inhibition and apoptosis of leukemic cells. Hence, we investigated the potent anti-leukemia activity of OTS167, a small molecule MELK kinase inhibitor, in AML, and found that the compound induced cell differentiation and apoptosis as well as decreased migration of AML cells. MELK expression was positively correlated with the expression of FOXM1 as well as its downstream target genes. Furthermore, MELK inhibition resulted in downregulation of FOXM1 activity and the expression of its downstream targets. Taken together, and given that OTS167 is undergoing a phase I clinical trial in solid cancer, our study warrants clinical evaluation of this compound as a novel targeted therapy for AML patients.

Asparaginase, which depletes asparagine and glutamine, activates amino acid stress response. Oxidative stress mediated by excessive reactive oxygen species (ROS) causes enhanced mitochondrial permeabilization and subsequent cell apoptosis and is considered a plausible mechanism for drug-induced hepatotoxicity, a common toxicity of asparaginase in adults with acute lymphoblastic leukemia (ALL). Studies investigating the pharmacogenetics of asparaginase in ALL are limited and focused on asparaginase-induced allergic reaction common in pediatric patients. Here, we sought to determine a potential association between the variant rs4880 in SOD2 gene, a key mitochondrial enzyme that protects cells against ROS, and hepatotoxicity during asparaginase-based therapy in 224 patients enrolled on CALGB-10102, a treatment trial for adults with ALL. We report that the CC genotype of rs4880 is associated with increased hepatotoxicity following asparaginase-based treatment. Thus, rs4880 likely contributes to asparaginase-induced hepatotoxicity, and functional studies investigating this SNP are needed to develop therapeutic approaches that mitigate this toxicity.

BackgroundT-cell-mediated rejection (TCMR) remains a major cause of kidney allograft failure. The characterization of T-cell repertoire in different immunological disorders has emerged recently as a novel tool with significant implications. We herein sought to characterize T-cell repertoire using next generation sequencing to diagnose TCMR.MethodsIn this prospective study, we analyzed samples from 50 kidney transplant recipients. We collected blood and kidney transplant biopsy samples at sequential time points before and post transplant. We used next generation sequencing to characterize T-cell receptor (TCR) repertoire by using illumina miSeq on cDNA synthesized from RNA extracted from six patients’ samples. We also measured RNA expression levels of FOXP3, CD8, CD4, granzyme and perforin in blood samples from all 50 patients.ResultsSeven patients developed TCMR during the first three months of the study. Out of six patients who had complete sets of blood and biopsy samples two had TCMR. We found an expansion of the TCR repertoire in blood at time of rejection when compared to that at pre-transplant or one-month post transplant. Patients with TCMR (n = 7) had significantly higher RNA expression levels of FOXP3, Perforin, Granzyme, CD4 and CD8 in blood samples than those with no TCMR (n = 43) (P = 0.02, P = 0.003, P = 0.002, P = 0.017, and P = 0.01, respectively).ConclusionsOur study provides a potential utilization of TCR clone kinetics analysis in the diagnosis of TCMR. This approach may allow for the identification of the expanded T-cell clones associated with the rejection and lead to potential noninvasive diagnosis and targeted therapies of TCMR.

Gain-of-function mutations of FLT3 (FLT3-ITD), comprises up to 30% of normal karyotype acute myeloid leukemia (AML) and is associated with an adverse prognosis. Current FLT3 kinase inhibitors have been tested extensively, but have not yet resulted in a survival benefit and novel therapies are awaited. Here we show that T-LAK cell-originated protein kinase (TOPK), a mitotic kinase highly expressed in and correlated with more aggressive phenotype in several types of cancer, is expressed in AML but not in normal CD34+ cells and that TOPK knockdown decreased cell viability and induced apoptosis. Treatment of AML cells with TOPK inhibitor (OTS514) resulted in a dose-dependent decrease in cell viability with lower IC50 in FLT3-mutated cells, including blasts obtained from patients relapsed after FLT3-inhibitor treatment. Using a MV4-11-engrafted mouse model, we found that mice treated with 7.5 mg/kg IV daily for 3 weeks survived significantly longer than vehicle treated mice (median survival 46 vs 29 days, P < 0.001). Importantly, we identified TOPK as a FLT3-ITD and CEBPA regulated kinase, and that modulating TOPK expression or activity resulted in significant decrease of FLT3 expression and CEBPA phosphorylation. Thus, targeting TOPK in FLT3-ITD AML represents a novel therapeutic approach for this adverse risk subset of AML.

Although recent progress in understanding the biology and optimizing the treatment of acute lymphoblastic leukemia (ALL) has improved cure rates of childhood ALL to nearly 90%, the cure rate in adult ALL remains less than 50%. The poor prognosis in adult ALL has in part been attributed to larger proportion of high-risk leukemia showing drug resistance. Thus, identifying novel therapeutic targets in ALL is needed for further improvements in treatment outcomes of adult ALL. Genetic aberration of chromatin-modifying molecules has been recently reported in subtypes of ALL, and targeting components of chromatin complexes has shown promising efficacy in preclinical studies. Suppressor of variegation 3-9 homologue 2 (SUV39H2), also known as KMT1B, is a SET-domain–containing histone methyltransferase that is upregulated in solid cancers, but its expression is hardly detectable in normal tissues. Here, we show that SUV39H2 is highly expressed in ALL cells but not in blood cells from healthy donors and also that SUV39H2 mRNA is expressed at significantly higher levels in bone marrow or blood cells from patients with ALL obtained at diagnosis compared with those obtained at remission (P = .007). In four ALL cell lines (Jurkat and CEM derived from T-ALL and RS4;11 and REH derived from B-ALL), SUV39H2 knockdown resulted in a significant decrease in cell viability (~ 77%, P < .001), likely through induction of apoptosis. On the other hand, SUV39H2 overexpression made cells more resistant to chemotherapy. We conclude that SUV39H2 is a promising therapeutic target and further investigation of this therapeutic approach in ALL is warranted.

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Tumor genomic profiling affected clinical management in a minority of patients with metastatic breast cancer, thus these data do not support the routine use of genomic profiling outside of a clinical trial.

Maternal embryonic leucine-zipper kinase (MELK), a member of the serine-threonine kinases snf1/AMP-activated protein family is involved in mammalian embryonic development. MELK is aberrantly upregulated in several types of solid cancer including glioblastoma and breast cancer, and implicated in formation and maintenance of cancer stem cells. Little is known about the relevance of this kinase in hematological malignancies. Our study aimed to explore the role of MELK in acute myeloid leukemia (AML) and identify whether targeting this kinase in leukemia stem cells may have therapeutic relevance. In order to characterize the expression of MELK in AML, we examined the protein levels in AML cell lines and primary blasts by western blot. MELK protein was expressed at variable levels in 10 out of 11 AML cell lines and primary blasts obtained from AML patients (n=5), representing diverse molecular and cytogenetic abnormalities of the disease. Importantly, we also found that MELK protein levels were higher in CD34+ cells compared with the more differentiated CD34- primary blasts from AML patient. To determine the role of MELK in AML, we utilized a loss of function approach in three AML cell lines (KG1, MV4-11 and U937 cells) and assessed cell viability by MTS. Cells transfected with MELK-siRNA showed significant decrease in cell viability (40-60%, P&lt;0.05) compared with cells transfected with control-siRNA. In addition, knockdown of MELK expression resulted in increased apoptosis in MV4-11 and U937 cells assessed by AnnexinV staining. OTS167, a small molecule MELK kinase inhibitor is currently undergoing phase I clinical testing in patients with advanced solid malignancies. To examine the anti-leukemia activity of OTS167 in AML, we treated nine AML cell lines with this compound and assessed cells viability by MTS assay. Decrease in cell viability following treatment with increasing doses of OTS167 was time and dose dependent. Variable sensitivities among AML cell lines (IC50∼10-40nM at 48hrs) were observed. In addition, the compound promoted cell differentiation as assessed by CD11b staining in U937 cells. Treatment with (25 and 50nM) of OTS167 induced apoptosis by ∼30 and 40% in MV4-11 and MOLM13 cells at 48 hours post treatment (P=0.01 and 0.05, respectively). Similarly, OTS167 cytotoxicity (IC50 ∼ 10-40nM; n=3) and induced apoptosis was observed in primary AML blasts. In conclusion, MELK is expressed in AML cell lines and AML blasts with higher levels observed in less differentiated cells. MELK knockdown results in growth inhibition, apoptosis, and cell death suggesting that this kinase may act as a potential therapeutic target. MELK kinase inhibitor (OTS167) causes cytotoxicity in AML cell lines and primary blasts. These results warrant further preclinical evaluation of this compound as a novel targeted therapy for patients with AML. Citation Format: Houda Alachkar, Martin Mutonga, Suyoun Chung, Yo Matsuo, Wendy Stock, Yusuke Nakamura. Preclinical efficacy of maternal embryonic leucine-zipper kinase (MELK) inhibition in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 952. doi:10.1158/1538-7445.AM2014-952