Loss of RAF kinase inhibitor protein is involved in myelomonocytic differentiation and aggravates RAS-driven myeloid leukemogenesis

Caraffini, Veronica; Geiger, Olivia; Rosenberger, Angelika; Hatzl, Stefan; Perfler, Bianca; Berg, Johannes Lorenz; Lim, Clarice X.; Strobl, Herbert; Kashofer, Karl; Schauer, Silvia; Beham‐Schmid, Christine; Höefler, Gerald; Geißler, Klaus; Quehenberger, Franz; Kölch, Walter; Athineos, Dimitris; Blyth, Karen; Wölfler, Albert; Sill, Heinz; Zebisch, Armin

doi:10.3324/haematol.2018.209650

Cited by 13 publications

(26 citation statements)

References 47 publications

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“…Primary AML patient specimens were collected at the stage of diagnosis and the stage of relapse or primary chemorefractory disease (R/R stage) at the Division of Hematology, Medical University of Graz, Graz, Austria (MUG). All samples were processed by Ficoll density gradient centrifugation, and mononuclear cell fractions were stored in the Biobank at the MUG until further use as described previously [51][52][53][54][55][56]. Additionally, cytospin preparations from mononuclear cell layers were analyzed, and only samples with a blast cell percentage of >80% were retained for further analyses.…”

Section: Patient Samples and Cell Linesmentioning

confidence: 99%

“…Stocks from low passages were frozen, and cells always were kept in culture for less than six months after thawing. Cells were authenticated regularly by a variable number of tandem repeat DNA profiling (VNTR) as described earlier [28,51,58,59]. The study was approved by the institutional review board of the Medical University of Graz (EK 30-464 ex 17/18), and informed consent was obtained from all individuals.…”

Section: Patient Samples and Cell Linesmentioning

confidence: 99%

“…All media were supplemented with 10% heat-inactivated FCS and 1× antibiotic-antimycotic, including 100 µg/mL streptomycin, 100 U/mL penicillin, and 0.25 µg/mL amphotericin B (Thermo Fisher Scientific, Waltham, MA, USA). Lentiviral transduction for overexpression of miR-23a-3p in THP-1 and U937 was performed using a psi-pEZX-MR03 expression construct (Genecopeia, Rockville, MD, USA) as previously described [28,51,53]. Stable selection and maintenance were performed in the presence of 1.0 µg/mL puromycin.…”

Section: Cell Culture Lentiviral Transduction and Transfectionmentioning

confidence: 99%

“…cDNA was synthesized from 1 µg RNA using the Taq Man®Reverse Transcription Reagents (Applied Biosystems, Foster City, CA, USA) for mRNA and miScript II Reverse Transcriptase Kit (Qiagen) for miRNA, respectively. Real-time quantitative PCR (qPCR) was performed using the ∆∆C t method, as previously described [51,53,[59][60][61][62]. B2M and GUSB were employed as control genes for mRNA analyses, whereas, RNU6 and SNORD44 were used for miRNA expression profiling.…”

Section: Qpcr and Immunoblot Analysismentioning

confidence: 99%

“…NB4 (for miRNA qPCR of the patient samples) and cells transfected with scrambled controls (for all other experiments), respectively, served as calibrators. Immunoblot was performed as described previously [51,53,59,61,62], using the following antibodies: Anti-TOP2B (#sc-365071; Santa Cruz, Dallas, TX, USA) and anti-β-Actin (#A5441; Sigma Aldrich, St. Louis, MO, USA).…”

Section: Qpcr and Immunoblot Analysismentioning

confidence: 99%

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Increased Expression of Micro-RNA-23a Mediates Chemoresistance to Cytarabine in Acute Myeloid Leukemia

Hatzl

Perfler

Wurm

et al. 2020

Cancers

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Resistance to chemotherapy is one of the primary obstacles in acute myeloid leukemia (AML) therapy. Micro-RNA-23a (miR-23a) is frequently deregulated in AML and has been linked to chemoresistance in solid cancers. We, therefore, studied its role in chemoresistance to cytarabine (AraC), which forms the backbone of all cytostatic AML treatments. Initially, we assessed AraC sensitivity in three AML cell lines following miR-23a overexpression/knockdown using MTT-cell viability and soft-agar colony-formation assays. Overexpression of miR-23a decreased the sensitivity to AraC, whereas its knockdown had the opposite effect. Analysis of clinical data revealed that high miR-23a expression correlated with relapsed/refractory (R/R) AML disease stages, the leukemic stem cell compartment, as well as with inferior overall survival (OS) and event-free survival (EFS) in AraC-treated patients. Mechanistically, we demonstrate that miR-23a targets and downregulates topoisomerase-2-beta (TOP2B), and that TOP2B knockdown mediates AraC chemoresistance as well. Likewise, low TOP2B expression also correlated with R/R-AML disease stages and inferior EFS/OS. In conclusion, we show that increased expression of miR-23a mediates chemoresistance to AraC in AML and that it correlates with an inferior outcome in AraC-treated AML patients. We further demonstrate that miR-23a causes the downregulation of TOP2B, which is likely to mediate its effects on AraC sensitivity.

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Section: Patient Samples and Cell Linesmentioning

confidence: 99%

Section: Patient Samples and Cell Linesmentioning

confidence: 99%

Section: Cell Culture Lentiviral Transduction and Transfectionmentioning

confidence: 99%

Section: Qpcr and Immunoblot Analysismentioning

confidence: 99%

Section: Qpcr and Immunoblot Analysismentioning

confidence: 99%

See 3 more Smart Citations

Increased Expression of Micro-RNA-23a Mediates Chemoresistance to Cytarabine in Acute Myeloid Leukemia

Hatzl

Perfler

Wurm

et al. 2020

Cancers

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miR-23a mediates resistance to hypomethylating agents in myeloid neoplasms

et al. 2021

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Comparison of acute myeloid leukemia and myelodysplastic syndromes with TP53 aberrations

et al. 2022

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TP53 aberrations are found in approximately 10% of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) and are considered early driver events affecting leukemia stem cells. In this study, we compared features of a total of 84 patients with these disorders seen at a tertiary cancer center. Clinical and cytogenetic characteristics as well as immunophenotypes of immature blast cells were similar between AML and MDS patients. Median overall survival (OS) was 226 days (95% confidence interval [CI], 131–300) for the entire cohort with an estimated 3-year OS rate of 11% (95% CI, 6–22). OS showed a significant difference between MDS (median, 345 days; 95% CI, 235–590) and AML patients (median, 91 days; 95% CI, 64–226) which is likely due to a different co-mutational pattern as revealed by next-generation sequencing. Transformation of TP53 aberrant MDS occurred in 60.5% of cases and substantially reduced their survival probability. Cox regression analysis revealed treatment class and TP53 variant allele frequency as prognostically relevant parameters but not the TP53-specific prognostic scores EAp53 and RFS. These data emphasize similarities between TP53 aberrant AML and MDS and support previous notions that they should be classified and treated as a distinct disorder.

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Loss of RAF kinase inhibitor protein is involved in myelomonocytic differentiation and aggravates RAS-driven myeloid leukemogenesis

Cited by 13 publications

References 47 publications

Increased Expression of Micro-RNA-23a Mediates Chemoresistance to Cytarabine in Acute Myeloid Leukemia

Increased Expression of Micro-RNA-23a Mediates Chemoresistance to Cytarabine in Acute Myeloid Leukemia

miR-23a mediates resistance to hypomethylating agents in myeloid neoplasms

Comparison of acute myeloid leukemia and myelodysplastic syndromes with TP53 aberrations

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