Association of relapse-linked ARID5B single nucleotide polymorphisms with drug resistance in B-cell precursor acute lymphoblastic leukemia cell lines

Abstract Background The genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents. Methods We genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment. Results No significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p=0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p=0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml). Conclusion These observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.

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Association of relapse-linked ARID5B single nucleotide polymorphisms with drug resistance in B-cell precursor acute lymphoblastic leukemia cell lines

10.21203/rs.3.rs-28763/v3 ◽

2020 ◽

Author(s):

MINORI TAMAI ◽

Meixian Huang ◽

Keiko Kagami ◽

Masako Abe ◽

Shinpei Somazu ◽

...

Keyword(s):

Gene Expression ◽

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Risk Allele ◽

Lymphoblastic Leukemia ◽

Chemotherapeutic Agents ◽

Nucleotide Polymorphisms ◽

Cell Precursor ◽

Single Nucleotide ◽

Ic50 Values

Abstract Background The genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents. Methods We genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment. Results No significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p=0.031 in Mann–Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p=0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml). Conclusion These observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.

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Association of relapse-linked ARID5B single nucleotide polymorphisms with drug resistance in B-cell precursor acute lymphoblastic leukemia cell lines

Cancer Cell International ◽

10.1186/s12935-020-01524-0 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Minori Tamai ◽

Meixian Huang ◽

Keiko Kagami ◽

Masako Abe ◽

Shinpei Somazu ◽

...

Keyword(s):

Drug Resistance ◽

Acute Lymphoblastic Leukemia ◽

Single Nucleotide Polymorphisms ◽

B Cell ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Nucleotide Polymorphisms ◽

Cell Precursor ◽

Single Nucleotide ◽

Leukemia Cell Lines

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Evaluation Of Sphingosine Kinase 1 As a Therapeutic Target In B-Lineage Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.1426.1426 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1426-1426

Author(s):

Craig T Wallington-Beddoe ◽

Stuart M Pitson ◽

Jason A Powell ◽

Kenneth F Bradstock ◽

Linda J Bendall

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Chemotherapeutic Agents ◽

Parp Cleavage ◽

Signalling Pathways ◽

Wide Range ◽

Ic50 Values

Abstract Sphingosine 1-phosphate (S1P) is a bioactive lipid with roles in cell proliferation and survival. S1P is produced by the sphingosine kinases, SphK1 and SphK2. SphK1 is over-expressed in a number of malignancies and evidence points overwhelmingly to a pro-survival role. Furthermore, SphK1 has been shown to correlate with the clinical outcome of certain tumors. Here we focus on SphK1 as an important oncogenic target in acute lymphoblastic leukemia (ALL). We have previously shown SphK1 protein to be over-expressed and activated (Ser225 phosphorylation) in ALL cell lines and primary patient samples compared to normal CD34+CD19+ B-cell progenitors. Furthermore, we have reported the importance of SphK1 in the development of ALL by transducing B-cell progenitors isolated from WT or SphK1-/- mice with the ALL associated p185 form of the oncogenic fusion gene BCR/ABL and injecting transduced cells into sub-lethally irradiated WT mice. The absence of SphK1 significantly reduced the incidence of ALL in recipient mice (ASH 2012). Inhibition of SphK1 by the selective inhibitor SK1-I significantly reduced intra-cellular S1P concentrations (p=0.017 and p=0.003 at 24 and 48 hours respectively) in 3 cell lines examined, indicating that the drug targets this enzyme. SK1-I killed ALL cells as determined by annexin V/PI flow cytometric analysis with IC50 values ranging from 12 µM to 18 µM at 72 hours. Furthermore, SK1-I induced cell death in primary patient ALL cells by 16 hours. This agent resulted in virtually no caspase-3 cleavage and cell death was not prevented by the pan-caspase inhibitor Z-VAD-FMK (p=0.45, n=4). Marked cytoplasmic vacuolation was detected by light microscopy, with LC3 processing present by Western blot, consistent with the development of autophagy. However, the autophagy inhibitor 3MA failed to prevent SK1-I-mediated cell death. These results suggest that the cell death associated with inhibition of SphK1 in ALL cells is caspase-independent and cannot be attributed to autophagy. Surprisingly, conventional chemotherapeutic agents such as doxorubicin and vincristine failed to synergize with SK1-I, however, synergistic killing was observed when SK1-I was combined with 500 nM imatinib over 72 hours in Philadelphia-positive (BCR/ABL+) ALL cells. We have developed a novel SphK1 inhibitor, MP8, that targets the enzyme via a different mechanism to SK1-I, since it blocks ATP binding. MP8 reduced intra-cellular S1P in Jurkat cells by 43% compared to untreated controls, and killed Jurkat and SUP-B15 cells over 24 to 48 hours with IC50 values of 8 µM. Additionally, MP8 induced cell death in primary patient ALL cells by 24 hours. This agent resulted in classic apoptotic cell death, which was rescued by Bcl-2 over-expression, resulting in near complete reversal of PARP cleavage. SphK1 has indisputable tumor-promoting properties and lies downstream of a number of signalling pathways known to be dysregulated in ALL. Here we show that SphK1 is over-expressed and activated in ALL cells and targeting SphK1 has potent cytotoxic effects in a wide range of cell lines and patient samples. Furthermore, genetic deletion of Sphk1 significantly reduced the incidence of murine BCR/ABL-driven ALL. These findings suggest further examination of the role SphK1 plays in ALL will uncover novel interactions with oncogenic signalling pathways and paves the way for the inclusion of SphK1 inhibitors in future pre-clinical trials. Disclosures: No relevant conflicts of interest to declare.

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Anti-Leukemia Activity of the Selective BCL-2 Inhibitor ABT-199 in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By MCL-1/BCL-2 Expression Serving As Biomarker for Treatment Response

Blood ◽

10.1182/blood.v128.22.1081.1081 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1081-1081

Author(s):

Felix Seyfried ◽

Salih Demir ◽

Rebecca Hörl ◽

Stefan Köhrer ◽

Annika Scheffold ◽

...

Keyword(s):

Cell Death ◽

Acute Lymphoblastic Leukemia ◽

Cell Lines ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

High Ratio ◽

Cell Precursor ◽

Free Survival ◽

Ic50 Values

Abstract Despite superior outcome and survival of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), relapse occurs in 10-20% and is associated with poor outcome, clearly indicating future challenges including reduction of relapse rates and effective treatment of reoccurred leukemia. Deficiencies in cell death and survival pathways have been implicated in therapy failure and treatment resistance in BCP-ALL. Members of the BCL-2 family are key regulators of these pathways and are therefore of interest as therapeutic targets. The small molecule ABT-199 binds selectively to BCL-2, inhibits its anti-apoptotic function and leads to release of pro-apoptotic molecules. Recently, ABT-199 has demonstrated clinical activity, particularly in poor prognosis CLL. However, insensitivity and resistance in different cases clearly emphasize the need of predictive markers for upfront identification of ABT-199 responsive leukemias. Here, we analyzed sensitivity for ABT-199 in a series of individual BCP-ALL samples, addressed mechanisms of resistance and evaluated markers indicating response to ABT-199. Anti-leukemic activities of ABT-199 were investigated in BCP-ALL cell lines (n=6) and patient-derived BCP-ALL primograft samples (n=17), which were established by transplantation of primary patient ALL cells obtained at diagnosis onto NOD/SCID mice. Half maximal inhibitory concentrations (IC50) for ABT-199 were analyzed for each sample. Expression of apoptosis regulating molecules was investigated by western blot analysis and associated with ABT-199 responsiveness. Two MCL-1 deficient ALL cell lines were generated by CRISPR/Cas9 gene editing. Leukemia free-survival of ALL bearing animals was analyzed after in vivoABT-199 treatment. The majority of BCP-ALL samples showed sensitivity for ABT-199 induced cell death in the nanomolar range, both in cell lines (n=4, IC50: 29 - 422 nM) and patient-derived primograft samples (n=10, IC50: 1.7 - 74 nM), while 2 cell lines and 7 primograft leukemias showed insensitivity with IC50 values above 1 µM. ABT-199 binds directly to BCL-2 and upon binding, pro-apoptotic Bcl-2 family molecules like Bim are dislocated from BCL-2 and induce apoptosis. The anti-apoptotic BCL-2 family member MCL-1 is not bound by ABT-199, but sequesters pro-apoptotic molecules dislocated from BCL-2 leading to interruption of apoptosis induction. Therefore, we addressed expression levels of BCL-2 and MCL-1. We found high BCL-2 levels in ABT-199 sensitive and low BCL-2 levels in resistant leukemia samples and an opposite pattern for MCL-1 (high in resistant and low MCL-1 in sensitive ALL), in line with previous reports. Most interestingly, a high ratio of MCL-1 to BCL-2 expression (high MCL-1, low BCL-2) was significantly associated with high IC50 values/resistance (Spearman Rho correlation, p= .01), whereas a low MCL-1/BCL-2 ratio indicated ABT-199 sensitivity. Two of the 6 cell lines showed ABT-199 resistance (IC50 > 1 µM) and high Mcl-1 expression. Effective MCL-1 knock-out in both cell lines led to a clear sensitization for ABT-199 with up to 40-fold reduced IC50 values, clearly indicating MCL-1 as a key mediator of ABT-199 resistance in BCP-ALL. Finally, we also evaluated the anti-leukemia activity of ABT-199 in a preclinical setting in vivo. Two patient-derived leukemias, one with a low MCL-1/BCL-2 ratio of 0.9 and the other with a high ratio of 16.1, indicative of ABT-199 sensitivity or resistance, were transplanted onto NOD/SCID mice and treated with ABT-199 for 10 days after ALL engraftment. Most interestingly, a significantly increased leukemia free survival was observed in ABT-199 as compared to vehicle treated recipients (p<0.001) of the leukemia with the low MCL-1/BCL-2 ratio, in contrast to similar survival times of vehicle or ABT-199 treated animals bearing the high MCL-1/BCL-2 ratio ALL, clearly showing the predictive value of BCL-2 and MCL-1 levels in BCP-ALL. Taken together, ABT-199 shows anti-leukemia activity in the majority of BCP-ALL samples, with a strong association of high BCL-2 and low MCL-1 levels with ABT-199 sensitivity. Silencing of MCL-1 clearly revealed a crucial role for MCL-1 as mediator of ABT-199 resistance. Importantly, in vivo evaluation of ABT-199 in a preclinical setting highlighted the predictive value of BCL-2/MCL-1 expression for the identification of patients who would benefit from future BCL-2 directed therapies. Disclosures Stilgenbauer: Genzyme: Consultancy, Honoraria, Other: Travel grants , Research Funding; Genentech: Consultancy, Honoraria, Other: Travel grants , Research Funding; Janssen: Consultancy, Honoraria, Other: Travel grants , Research Funding; Sanofi: Consultancy, Honoraria, Other: Travel grants , Research Funding; Hoffmann-La Roche: Consultancy, Honoraria, Other: Travel grants , Research Funding; Novartis: Consultancy, Honoraria, Other: Travel grants , Research Funding; GSK: Consultancy, Honoraria, Other: Travel grants , Research Funding; Gilead: Consultancy, Honoraria, Other: Travel grants , Research Funding; Pharmacyclics: Consultancy, Honoraria, Other: Travel grants , Research Funding; AbbVie: Consultancy, Honoraria, Other: Travel grants, Research Funding; Mundipharma: Consultancy, Honoraria, Other: Travel grants , Research Funding; Celgene: Consultancy, Honoraria, Other: Travel grants , Research Funding; Amgen: Consultancy, Honoraria, Other: Travel grants, Research Funding; Boehringer Ingelheim: Consultancy, Honoraria, Other: Travel grants , Research Funding.

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