scholarly journals HOXA11 Regulates Chemoresistance By Modulating p53 Gene Expression in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5182-5182
Author(s):  
Xutao Guo ◽  
Bowen Yan ◽  
Yi Qiu
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
P53 Gene ◽  
Resistant Cell ◽  
Wild Type ◽  
P53 Expression ◽  
Acute Myeloid ◽  
Resistant Cells

Acute myeloid leukemia (AML) exhibits large intrinsic variation in drug responsiveness due to its inherent heterogeneity. Therefore, it is important to understand the resistant mechanism in order to improve the treatment. In our previously study, the OCI-AML2-resistant cell lines were established to resist cytarabine (Ara-C) in the concentration of 50 µM (OCI-AML2 R50). The RNA-seq results showed that many genes changed in the resistant cells compared to wild type OCI-AML2 cells. One of the most remarkably decreased gene in resistant cells was HOXA11 (Homeobox A11). It is the part of the A cluster on chromosome 7 and encodes a DNA-binding transcription factor which regulates gene expression, morphogenesis, and differentiation. In this study, we have evaluated the importance of HOXA11 in AML chemoresistance. We found that knockdown of HOXA11 repressed the WT OCI-AML2 cell proliferation and increased the population of cells expressing CD123 and CD47 LSC (Leukemia stem cell) markers and enhanced the resistance to Ara-C in vitro, while overexpression of HOXA11 showed the reverse effect. These results support the idea that HOXA11 promotes drug sensitivity and apoptosis in AML. However, the result also showed that overexpression of HOXA11 repressed the OCI-AML2 R50 cell proliferation and enhanced the resistance. Therefore, HOXA11 plays opposite role in sensitive cells and resistant cells. We further investigated the mechanism for these effects. We found that knockdown of HOXA11 decreased the p53 gene expression and overexpression of HOXA11 increased the expression of p53 in OCI-AML2 and R50 cells. Further, in OCI-AML2 R50 cells p53 has a hotspot mutation in DNA binding site and studies have shown that p53 mutation enhance cancer cell survival and chemoresistance. Therefore, our study shows dual roles for HOXA11 in cell survival. In p53 wild type parental AML2 cells, HOXA11 induces wild type p53 expression to enhance drug sensitivity while in resistant cell, HOXA11 promotes mutant p53 expression and enhances the resistance of chemotherapy. Disclosures No relevant conflicts of interest to declare.

Activation of the Wnt/β-Catenin Pathway Mediates Lenalidomide Resistance in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 113-113 ◽  
Author(s):  
Chad C. Bjorklund ◽  
Deborah J. Kuhn ◽  
Jairo A. Matthews ◽  
Michael Wang ◽  
Veerabhadran Baladandayuthapani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Myeloma ◽  
Cell Lines ◽  
Expression Profiling ◽  
Fold Increase ◽  
Resistant Cell ◽  
Refractory Disease ◽  
Wild Type ◽  
Myeloma Cells ◽  
Resistant Cells

Abstract Abstract 113 Background: Novel drugs such as the immunomodulatory agent lenalidomide have revolutionized the treatment of multiple myeloma, as evidenced by an increasing overall survival for patients with both newly-diagnosed, and relapsed and/or refractory disease. Despite these improvements, myeloma remains incurable, and is still characterized by a trend for increasing chemoresistance at relapse, with a decreasing duration of benefit from each successive line of therapy. By understanding the mechanisms responsible for the emergence of drug resistance, which have so far not been well characterized in the case of lenalidomide, it may be possible to rationally design novel regimens that could either overcome this resistance, or possibly prevent its emergence altogether. Methods: To improve our understanding of the mechanisms responsible for lenalidomide resistance, we developed cell line models of interleukin (IL)-6-dependent (ANBL-6 and KAS-6/1) and –independent (U266 and MM1.S) lenalidomide-resistant multiple myeloma cells. Starting at a concentration that was 1/10 of the IC50 for lenalidomide's anti-proliferative effects in drug-naïve cells, increasing drug concentrations were used until all the cell lines could proliferate and maintain cell membrane integrity in the presence of 10 μM lenalidomide. These cell lines were then used as an in vitro model of lenalidomide-specific drug resistance, and subjected to further characterization, including with gene expression profiling. Results: Resistance to lenalidomide was evidenced by a dramatic, 100-1000-fold increase in the IC50 values of these myeloma cells. In the case of ANBL-6 cells, for example, drug-naïve cells showed an IC50 of 0.14 μM using tetrazolium dye-based viability assays, but this increased to >100 μM in the drug-resistant cells, as was the case in U266 and MM1.S cells. This resistance was a stable phenotype, since removal of lenalidomide for seven to ninety days from cell culture conditions did not re-sensitize them when 10 μM lenalidomide was reintroduced. Gene expression profiling followed by pathway analysis to examine changes at the transcript level between wild-type parental and lenalidomide-resistant cell lines identified the Wnt/β-catenin pathway as the most altered across all cell lines. Increased expression was seen in several members of the low-density-lipoprotein receptor related protein family, including LRP1 and 5; members of the wingless-type MMTV integrations site family, including WNT3 and 4; β-catenin; and downstream Wnt/β-catenin targets such as CD44. Similar changes were detected in primary samples from a patient who developed clinically lenalidomide-refractory disease. Reporter assays revealed an up to 5-fold increase in LEF/TCF-dependent transcription both in drug-naïve cells acutely exposed to lenalidomide, and in their chronically exposed, lenalidomide-resistant clones. Western blotting and flow cytometry confirmed that these lenalidomide-resistant cells had increased expression by 2-20 fold of β-catenin and CD44, as well as other LEF/TCF targets, including Cyclin D1 and c-Myc. Comparable changes occurred after lenalidomide exposure in myeloma cells grown in the context of bone marrow stroma. Notably, lenalidomide-resistant cells showed decreased expression of casein kinase 1 and increased phosphorylation of glycogen synthase kinase 3 at Ser21/9, both of which would reduce the phosphorylation of β-catenin needed for its later proteasome-mediated degradation. Stimulation of the Wnt/β-catenin pathway with recombinant human Wnt3a resulted in resistance to lenalidomide in wild-type, drug-naïve cells, as evidenced by a 10-fold increase in the IC50. Conversely, exposure of lenalidomide-resistant cell lines to quercetin, a known antagonist of the β-catenin/TCF interaction, induced a partial re-sensitization to lenalidomide. Conclusions: These data support the hypothesis that activation of the Wnt/β-catenin pathway represents a mechanism of both acute and chronic resistance to the anti-proliferative effects of lenalidomide in multiple myeloma. Moreover, they support the development of strategies aimed at suppressing Wnt/β-catenin activity to resensitize multiple myeloma to the effects of this immunomodulatory agent in vivo. Disclosures: No relevant conflicts of interest to declare.

High p53 Protein Expression LEVEL Independent Of Mutational Status Is An Adverse Prognostic Factor For Survival In ACUTE Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1330-1330
Author(s):  
Alfonso Quintas-Cardama ◽  
Sean M. Post ◽  
Kensuke Kojima ◽  
Yi Hua Qiu ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mutant P53 ◽  
P53 Mutations ◽  
Wild Type ◽  
P53 Pathway ◽  
P53 Expression ◽  
Mutational Status ◽  
Wild Type P53 ◽  
Acute Myeloid

Abstract Background The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML), particularly in cases with high-risk cytogenetics. It has been shown that p53 stabilization, which frequently occurs when the protein is mutated, can compromise its function. We have shown that p53 stabilization, regardless of the presence of mutations, suggesting alterations of other components in the p53 pathway. Methodology p53 expression was determined using high-throughput reverse phase protein array (RPPA) technology in 719 samples from 511 pts. Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status of p53 alleles was assessed by Sanger sequencing of exons 5 through 9. Expression of components of the p53 pathway was determined using standard immunohistochemical techniques. Nutlin-3a was used in in vitro culture experiments. Results Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). p53 expression correlated directly with CD34 (p=0.001) and inversely correlated with WBC (p=0.007), PB and BM blast burden (p=0.0001), and survival (p=0.01). High p53 (p53high) expression was more associated with unfavorable cytogenetics, particularly -5 (p=0.00001). p53high resulted in lower complete remission (CR) rates (51% vs 56%; p=??), higher relapsed rates (82% vs 62%; p=??), and shorter median overall survival (OS; 29.8 vs. 51 wks, p=0.009) compared to p53low pts. Most cases with p53high had unfavorable cytogenetics. We next correlated p53 stabilization with the presence of p53 mutations in 68 pts. p53 mutations were detected in 20/54 (37%) p53high pts and in 0/14 (0%) pts with p53low. p53high, either in the presence (29 wks) or in the absence (24 wks) of p53 mutations (p=1.0), was associated with significantly shorter OS compared with p53low pts (56 wks; p=0.05). Multivariate analysis revealed p53 expression to be an independent risk factor for survival in AML (p=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), BAD (p=0.0001), cleaved PARP (p=0.002), and cleaved PARP (p=0.01), and negatively with p21 (p=0.01), and MDM2 (p=0.001).Given the similar OS in p53high pts carrying mutant or wild-type p53, we scored the immunohistochemical expression of MDM2, MDM4, and p21 in 30 p53high pts (9 p53 mutated, 21 wild-type p53). Overexpression of MDM2 was observed in 44% vs 48% pts with mutant vs wild-type p53, respectively, whereas rates were 67% vs 62% for MDM4, and 0% vs 19% for p21, for each respective genotype. Overall, of the 21 p53high pts carrying wild-type p53, 15 (71%) had overexpression of MDM2 and/or MDM4, whereas 81% had no p21 expression, indicating deficient activation of the p53 pathway similar to those cases carrying mutant p53. We are currently assessing response to nutlin-3a therapy in 24 primary AML samples (4 mutant p53, 20 wild-type p53). Results showing the impact of p53 mutation and/or stabilization, and expression levels of MDM2, MDM4, and p21 on nutlin-3a therapy will be presented. Conclusions p53 stabilization (p53high) is a powerful predictive and prognostic factor in AML, which is independent of the presence of mutant p53 alleles. Poor outcomes in pts with p53high lacking p53 mutations are very frequently associated with overexpression of negative regulators of p53 such as MDM2 and/or MDM4 and p21 downregulation, indicating a functionally altered p53 pathway. These findings may have implications for therapies targeting the MDM2/p53 axis in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Genomic markers of midostaurin drug sensitivity in FLT3 mutated and FLT3 wild-type acute myeloid leukemia patients

Oncotarget ◽  
2020 ◽  
Vol 11 (29) ◽  
pp. 2807-2818
Author(s):  
Mara W. Rosenberg ◽  
Kevin Watanabe-Smith ◽  
Jeffrey W. Tyner ◽  
Cristina E. Tognon ◽  
Brian J. Druker ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Wild Type ◽  
Genomic Markers ◽  
Acute Myeloid

scholarly journals Dasatinib Inhibits FLT3/ITD and PTPN11mutated Acute Myeloid Leukemia Cells Overexpressing SRC Tyrosine Kinases

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1451-1451
Author(s):  
Sigal Tavor ◽  
Tali Shalit ◽  
Noa Chapal Ilani ◽  
Yoni Moskovitz ◽  
Nir Livnat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Advisory Committees ◽  
Acute Myeloid

Background: Recent advances in acute myeloid leukemia(AML) targeted therapy improve overall survival. While these targeted therapies can achieve prolonged remissions, most patients will eventually relapseunder therapy. Our recent studies suggest that relapse most often originates from several sub-clones of leukemic stem cells (LSCs), present before therapy initiation, and selected due to several resistance mechanisms. Eradication of these LSCs during treatment induction /remission could thus potentially prevent relapse. The overall goal of the current study was to identify drugs which can be safely administrated to patients at diagnosis and that will target LSCs. Since simultaneously testing multiple drugs in vivo is not feasible, we used an in vitrohigh throughput drug sensitivity assay to identify new targets in primary AML samples. Methods: Drug sensitivity and resistance testing (DSRT) was assessed in vitro (N=46 compounds) on primary AML samples from patients in complete remission (N=29). We performed whole exome sequencing and RNAseq on samples to identify correlations between molecular attributes and in vitro DSRT. Results:Unsupervised hierarchical clustering analysis of in vitro DSRT, measured by IC50, identified a subgroup of primary AML samples sensitive to various tyrosine kinase inhibitors (TKIs). In this subgroup, 52% (9/17) of AML samples displayed sensitivity to dasatinib (defined as a 10-fold decrease in IC50 compared to resistant samples). Dasatinib has broad TKI activity, and is safely administered in the treatment of leukemia. We therefore focused our analysis on predicting AML response to dasatinib, validating our results on the Beat AML cohort. Enrichment analysis of mutational variants in dasatinib-sensitive and resistant primary AML samples identified enrichment of FLT3/ITD (p=0.05) and PTPN11(p=0.05) mutations among dasatinib responders. Samples resistant to dasatinib were enriched with TP53 mutations (p=0.01). No global gene expression changes were observed between dasatinib-sensitive and resistant samples in our cohort, nor in the Beat AML cohort. Following this, we tested the differential expression of specific dasatinib-targeted genes between dasatinib-responding and resistant samples. No significant differences were identified. However, unsupervised hierarchical clustering of dasatinib targeted genes expression in our study and in the Beat AML cohort identified a subgroup of AML samples (enriched in dasatinib responders) that demonstrated overexpression of three SRC family tyrosine kinases:FGR, HCK and LYN as well as PTK6, CSK, GAK and EPHB2. Analysis of the PTPN11 mutant samples revealed that the IC50 for dasatinib in 23 carriers of the mutant PTPN11 was significantly lower compared to the IC50 of PTPN11 wild type samples (p=0.005). LYN was also upregulated (p<0.001) in the mutant samples. We therefore hypothesized that gene expression of dasatinib-targeted genes could be used as a predictive biomarker of dasatinib response among FLT3/ITD carriers. We found that among FLT3/ITD AML carriers in the Beat AML cohort LYN, HCK, CSK and EPHB2 were significantly over-expressed in the dasatinib responding samples (N=27) as compared to the dasatinib resistant samples (N=35). To predict response to dasatinib among FLT3/ITD carriers we used a decision tree classifier based on the expression levels of these four genes. Our prediction model yielded a sensitivity of 74% and specificity of 83% for differentiating dasatinib responders from non-responders with an AUC of 0.84. Based on our findings, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. We found that in a subset of these samples, dasatinib significantly inhibited LSCs engraftment. This subset of FLT3/ITD AML samples expressed higher levels of LYN, HCK,FGR and SRC as compared to the FLT3/ITD samples that were not sensitive to dasatinib therapy in vivo. In summary, we identified a subgroup of AML patients sensitive to dasatinib, based on mutational and expression profiles. Dasatinib has anti-leukemic effects on both blasts and LSCs. Further clinical studies are needed to demonstrate whether selection of tyrosine kinase inhibitors, based on specific biomarkers, could indeed prevent relapse. Disclosures Tavor: Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS companies: Membership on an entity's Board of Directors or advisory committees.

A Functional Wild-Type p53 Gene Is Expressed in Human Acute Myeloid Leukemia Cell Lines

Blood ◽  
1998 ◽  
Vol 92 (8) ◽  
pp. 2977-2979 ◽  
Author(s):  
Trenna Sutcliffe ◽  
Loning Fu ◽  
Jacinth Abraham ◽  
Homayoun Vaziri ◽  
Samuel Benchimol
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
P53 Gene ◽  
Wild Type ◽  
Leukemia Cell Lines ◽  
Human Acute Myeloid Leukemia ◽  
Acute Myeloid ◽  
Myeloid Leukemia Cell

Double, but Not Single, CEBPA mutations Define a Subgroup of Acute Myeloid Leukemia with Favorable Outcome and a Distinct Gene Expression Profile

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 141-141 ◽  
Author(s):  
Bas J. Wouters ◽  
Claudia A.J. Erpelinck-Verschueren ◽  
Bob Lowenberg ◽  
Peter J.M. Valk ◽  
Ruud Delwel
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Multivariable Analysis ◽  
Favorable Outcome ◽  
Heterozygous Mutation ◽  
Wild Type ◽  
Mutation Status ◽  
Cebpa Mutations ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease characterized by various cytogenetic and molecular abnormalities, some of which can be used as prognostic markers. Mutations in the transcription factor CCAAT/enhancer binding protein alpha (CEBPA) occur in 5–10% of AML and have consistently been associated with a favorable outcome. Three types of mutations have been described: N-terminal out-of-frame mutations, inframe mutations in the basic leucine zipper (bZIP) region, and a small variable group of remaining aberrations. Most CEBPA mutant AML cases carry two mutations, usually on different alleles (double mutant). However, there are also cases that only express a single heterozygous mutation, and thus retain a wild type allele (single mutant). It is not known whether single and double CEBPA mutations should be considered of equal biological and/or clinical importance. We applied dHPLC WAVE technology in combination with nucleotide sequencing of the entire CEBPA gene in a cohort of 598 cases of adult de novo AML. After exclusion of previously described polymorphisms, we identified 41 cases (6.9%) with at least one mutation. Of these cases, 28 carried double mutations, i.e. two different heterozygous mutations or one homozygous mutation, whereas the remaining cases carried a single heterozygous mutation. To investigate whether CEBPA mutations were associated with specific transcriptional signatures, we examined genome-wide gene expression (GEP) data of 525/598 AMLs, including 38/41 CEBPA mutant cases. Class prediction of total CEBPA mutation status based on GEP data resulted in a relatively large number of false negatives in cross-validation using the PAM algorithm (sensitivity 68%, specificity 99%). Strikingly however, all these missed cases appeared to be of the single mutant group, while the double mutants were recognized with high accuracy. In agreement, unsupervised cluster analysis of the 525 AMLs led to distinct grouping of cases with double mutations, while cases with a single heterozygous mutation did not. These observations suggested that double and single CEBPA mutant AMLs represent distinct biological entities. We next assessed the clinical relevance of this finding. In concordance with previous studies, total CEBPA mutation status associated with favorable overall survival (OS) and event-free survival (EFS) (P=0.023 and P=0.042, log rank test), which was maintained in multivariable Cox’s proportional hazards models with cytogenetic risk group, FLT3-ITD and NPM1 mutation status, age and white blood cell count (hazard ratio [HR] 0.47, 95% confidence interval [CI] 0.29–0.77; P=0.002 and HR 0.52, 95% CI 0.33–0.82; P=0.004). Surprisingly, when the double and single CEBPA mutant cases were separately analyzed, only the double mutants showed a highly favorable outcome, while the single mutants could not be distinguished from CEBPA wild type AMLs (P=0.003 versus P=0.51 (OS) and P=0.004 versus P=0.18 (EFS)). In multivariable analysis, CEBPA double mutation status remained associated with favorable outcome (OS HR 0.31, 95% CI 0.16–0.59; P<0.001, and EFS HR 0.34; 95% CI 0.19–0.61; P<0.001), contrasting with the single mutants (HR 1.18, 95% CI 0.58–2.41; P=0.64 and HR 1.65, 95% CI 0.84–3.23; P=0.15). Similarly, in multivariable analysis in the selected normal karyotype subset (n=193), CEBPA double mutations, but not single mutations, were significantly associated with OS (P=0.026 versus P=0.24) and EFS (P=0.013 versus P=0.42). In conclusion, these data demonstrate the existence of distinct transcriptional and clinical characteristics of AML cases with double CEBPA mutations and imply that it is crucial to discriminate them from single mutants to identify those patients with a favorable prognosis.

Anti-Leukemia Effect of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2756-2756
Author(s):  
Hui Yang ◽  
Zachary S. Bohannan ◽  
Zhihong Fang ◽  
Irene Gañán-Gómez ◽  
Yue Wei ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Resistant Cell ◽  
Nucleotide Synthesis ◽  
Cell Numbers ◽  
Induced Apoptosis ◽  
Dose Dependent ◽  
Acute Myeloid

Abstract Introduction Inosine 5'- monophosphate dehydrogenase (IMPDH) plays a critical role in nucleotide synthesis by serving as a rate-limiting step for the de novo production of guanine from its precursors. Overexpression of IMPDH has been observed in both solid and hematologic malignancies. FF-10501-01 is a potent new competitive IMPDH inhibitor. In this study, we systematically investigated the anti-leukemia effect of FF-10501-01 in acute myeloid leukemia (AML) cell lines, including hypomethylating agent (HMA)-resistant derivative cells. Methods Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937) as well as three other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle status. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentration of guanine nucleotides, with mycophenolic acid (MPA) treated cells used as positive control. We also studied the effect of guanosine supplementation on FF-10501-01-treated cells. Results To understand whether FF-10501-01 was able to effectively limit AML cell proliferation, we subjected a variety of cell lines to 72 hours of FF-10501-01 treatment at various concentrations. We also included a large number of HMA-resistant cell lines in an effort to understand whether or not FF-10501-01 could be a useful secondary or complimentary treatment to HMA therapy. FF-10501-01 inhibited the proliferation of all 13 AML cell lines studied with 72 hours of treatment. The IC-50 of FF-10501-01 ranged between 4.3 and 144.5 µM. The IC50 values for HMA-resistant cells were all higher than those values in their HMA-sensitive counterparts, except SKM1, in which the HMA-sensitive line had a higher IC50 than the HMA-resistant SKM1 line. To further understand the mechanism by which FF-10501-01 effectively reduced cell numbers in these cell lines, we assessed the level of apoptosis in each line after FF-10501-01 exposure. FF-10501-01-induced apoptosis was observed in all of the studied cell lines in a dose-dependent manner except for the HMA-resistant TF-1 cell line. We then assessed whether FF-10501-01 affected cell cycle progression. This effect was highly variable. Increased numbers of cells in G1 phase and decreased numbers of cells in S phase were observed in MOLM13, SKM1, and TF-1 cell lines treated with less than 100 µM FF-10501-01. To understand the mechanistic effects of FF-10501-01, we performed rescue experiments with both HMA-resistant and HMA-sensitive MOLM13 and HL60 cells. Concurrent treatment with FF-10501-01 and guanosine in these cells partially rescued the antiproliferation effect of FF-10501-01. To further characterize the effect of FF-10501-01 on guanosine metabolism, we then performed HPLC experiments to analyze the levels of phosphoguanosine in treated MOLM13 and SKM1 cells. FF-10501-01 treatment effectively reduced the intracellular phosphoguanosine levels in both cell lines. This effect was seen for GMP, GDP, and GTP. We then sought to assess whether the combination of FF-10501-01 and HMAs could be effective in limiting MOLM13 and HL-60 cell proliferation, especially in their HMA resistant derivatives. The combination of HMA and FF-10501-01 showed little synergy beyond the effects of FF-10501-01 alone, regardless of HMA sensitivity, except in HMA-resistant HL-60 cells, in which FF-10501-01 showed moderate synergy with HMA. We further assessed the antiproliferative effect of FF-10501-01 in bone marrow blast samples taken from 3 AML patients. There was a minor dose-dependent antiproliferation effect seen in these samples, but this was not statistically significant. Notably, one patient showed a sharp increase in cell counts at the lowest concentration of FF-10501-01, but that sample's cell numbers decreased more rapidly as FF-10501-01 concentration increased. This implies that FF-10501-01 treatment response may be related to cell proliferation. Conclusions The IMPDH inhibitor FF-10501-01 can produce potent anti-proliferative and apoptotic induction effects on AML cell lines, including HMA-resistant cell lines, through inhibition of de novo guanine nucleotide synthesis. These results indicate that FF-10501-01 might be a promising new therapeutic agent for AML. Disclosures Paradiso: Strategia Therapeutics, Inc.: Employment. Iwamura:FUJIFILM Corporation: Employment.

The Relationship between Mutation in HOXB1 Gene and Acute Myeloid Leukemia

2019 ◽  
Author(s):  
Mohammad Yahya Vahidi Mehrjardi ◽  
Seyed Mohsen Aghaei Zarch ◽  
Mohammadreza Dehghani
Keyword(s):  
Gene Expression ◽  
Experimental Study ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Rna Extraction ◽  
Expression Level ◽  
Wild Type ◽  
Significant Difference ◽  
Heterozygous Carriers ◽  
Acute Myeloid

Background: HOX genes are an exceedingly preserved family of homeodomain-involving transcription factors. They are related to a number of malignancies, comprising acute myeloid leukemia (AML). This study aimed to evaluate the effect of HOXB1 7bp deletion mutation on HOXB1gene expression in 36 individuals. Materials and Methods: The present cross-sectional study was done on a large Iranian family. In this experimental study, 5 homozygous 7bp deletion individuals along with their unaffected siblings and their parents were investigated. The candidate gene, HOXB1 was screened and analyzed in blood samples of these participants. After RNA extraction, cDNA was synthesized according to manufacturer’s protocol. HOXB1 expression level was analyzed by 2ΔΔCT method. All laboratory procedures used in this experimental study were carried out in genetic laboratory of Shahid Sadoughi University of Medical Sciences. Results: Sequence analysis of HOXB1 gene by ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) revealed a family with 5 homozygous (22±17 years) and 22 healthy heterozygous carriers (42±19 years) for 7bp deletion in HOXB1 gene along with 9  healthy wild type (55±41 years). Gene expression analysis by RT-qPCR demonstrated that expression level of HOXB1 gene in wild type and heterozygous carriers specimens had similar levels (p=0.05). Conclusion: Although HOXB1 mutations has been reported in AML, but association between HOXB1 mutation and AML was not found in our study. Additionally, HOXB1 expression levels showed no significant difference between wild type and heterozygous carriers. So, HOXB1 gene expression cannot provide a powerful tool to differentiate wild type from heterozygous carries.

scholarly journals Prognostic Significance of Expression of a Single MicroRNA,miR-181a, in Cytogenetically Normal Acute Myeloid Leukemia: A Cancer and Leukemia Group B Study

2010 ◽  
Vol 28 (36) ◽  
pp. 5257-5264 ◽  
Author(s):  
Sebastian Schwind ◽  
Kati Maharry ◽  
Michael D. Radmacher ◽  
Krzysztof Mrózek ◽  
Kelsi B. Holland ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Myeloid Leukemia ◽  
Prognostic Significance ◽  
Comprehensive Cancer Center ◽  
Wild Type ◽  
The Impact ◽  
Acute Myeloid

PurposeTo evaluate the prognostic significance of expression levels of a single microRNA, miR-181a, in the context of established molecular markers in cytogenetically normal acute myeloid leukemia (CN-AML), and to gain insight into the leukemogenic role of miR-181a.Patients and MethodsmiR-181a expression was measured in pretreatment marrow using Ohio State University Comprehensive Cancer Center version 3.0 arrays in 187 younger (< 60 years) adults with CN-AML. Presence of other molecular prognosticators was assessed centrally. A gene-expression profile associated with miR-181a expression was derived using microarrays and evaluated by Gene-Ontology analysis.ResultsHigher miR-181a expression associated with a higher complete remission (CR) rate (P = .04), longer overall survival (OS; P = .01) and a trend for longer disease-free survival (DFS; P = .09). The impact of miR-181a was most striking in poor molecular risk patients with FLT3-internal tandem duplication (FLT3-ITD) and/or NPM1 wild-type, where higher miR-181a expression associated with a higher CR rate (P = .009), and longer DFS (P < .001) and OS (P < .001). In multivariable analyses, higher miR-181a expression was significantly associated with better outcome, both in the whole patient cohort and in patients with FLT3-ITD and/or NPM1 wild-type. These results were also validated in an independent set of older (≥ 60 years) patients with CN-AML. A miR-181a-associated gene-expression profile was characterized by enrichment of genes usually involved in innate immunity.ConclusionTo our knowledge, we provide the first evidence that the expression of a single microRNA, miR-181a, is associated with clinical outcome of patients with CN-AML and may refine their molecular risk classification. Targeted treatments that increase endogenous levels of miR-181a might represent novel therapeutic strategies.

scholarly journals Metabolic Alterations May Contribute to Cabozantinib Resistance in Acute Myeloid Leukemia Cells with FLT3-ITD

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2785-2785
Author(s):  
Fang-Yu Lo ◽  
Kit Man Ng ◽  
Wen-Chun Chen ◽  
Chung-Yi Hu ◽  
Hsin-An Hou ◽  
...  
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Lactate Production ◽  
Resistant Cell ◽  
Atp Content ◽  
Rna Seq ◽  
Metabolic Alterations ◽  
Gapdh Activity ◽  
Acute Myeloid ◽  
Resistant Cells

Abstract Background: Internal tandem duplication in the juxtamembranal region of FLT3 gene (FLT3-ITD) is one of the most common mutations in acute myeloid leukemia (AML), resulting in constitutive activation of FLT3 signaling pathway. Therefore FLT3 have been proved to be as a useful target for AML treatment. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor (TKI), could selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Recently, cabozantinib was reported to be well tolerated in AML patients with FLT3-ITD and potentially be useful in the treatment of AML with FLT3-ITD. However, it is known that TKI-resistance in AML often cause higher relapse rates and lower survival rates. In order to study the drug resistance mechanism, we established cabozantinib-resistant cell lines from MV4-11 and Molm13 cells, after gradual escalating concentration of cabozantinib incubation, with increasing IC50 from 9.5nM to 1.5μM and from 1.06nM to 473.36nM, respectively. The cabozantinib-resistance cell lines were named MV4-11 XR and Molm-13 XR, respectively. Aims: To elucidate the mechanism of survival advantage of cabozantinib-resistance of MV4-11-XR and Molm13-XR. Materials and Methods : The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape recourse and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses were performed to predict the biological functions of DEGs. Quantitative PCR (Q-PCR) were used to validate the RNA-seq results. The extracellular acidification rate (ECAR) was measured using Seahorse bio-analyzer. In addition, to investigate the mitochondrial metabolism, analyses of oxygen consumption rate (OCR), glucose uptake, GAPDH activity, lactate production, ATP content were performed. Results: The gene expression profile in MV4-11 cells and Molm13 cells were used as baselines to establish the up- or down-regulated genes in MV4-11-XR and Molm13-XR cells, respectively. The FPKM were estimated with the selection criteria of q value<0.05 and [log2 (fold change0)>1 or <1 for significantly differential expression for up- and down-regulation, respectively. We identified a total of 1113 DEGs between the MV4-11 and MV4-11-XR cells, and a total of 1057 DEGs between the Molm13 and Molm13-XR cells. By using KEGG Mapper to interrogate pathways significance, we found that the metabolic pathway was the most significant in both up- and down-regulated DEGs in both resistant cells (MV4-11-XR up: 30 DEGs, down: 52 DEGs; Molm13-XR up: 27 DEGs, down: 58 DEGs). The information suggests that metabolic alterations occur in both drug resistant cell lines. Both MV4-11-XR and Molm13-XR cells showed higher glucose uptake, GAPDH activity, lactate production and ATP content than corresponding parental cells. Consistent with increased lactate export, analysis of glycolytic function showed a significant increase in glycolysis, glycolytic capacity and glycolytic reserve in both resistant cells. In addition, we showed increased basal mitochondrial and ATP-coupled respiration in both resistant cells, compared to their corresponding parental cells. Finally, decreased OCR / ECAR ratios indicated the relatively higher reliance on glycolysis in both resistant cells: 15.79 in Molm13-XR cells compared to 24.93 in Molm-13 cells, and 1.75 in MV4-11-XR cells compared to 9.97 in MV4-11 cells. Conclusion: Our study highlights that alteration of metabolic pathways may contribute cabozantinib resistance. We suggest that targeting these pathways may be a viable strategy to overcome cabozantinib resistance. Disclosures No relevant conflicts of interest to declare.

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