Functional identification of secondary mutations inducing autonomous growth in synergy with a truncated interleukin-3 receptor

Prassolov, Vladimir; Meyer, Johann; Brandenburg, Gunda; Hannemann, Jürgen; Bergemann, Jörg; Ostertag, Wolfram; Stocking, Carol

doi:10.1016/s0301-472x(01)00648-8

Cited by 6 publications

(7 citation statements)

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“…Withdrawal of IL-3 serves as a selective pressure to enrich for acquired mutations, but our data suggest these mutations exist prior to factor withdrawal. In contrast to earlier findings [32], we find no evidence that the time spent in culture between viral infection and factor withdrawal influences transformation rate. We recommend that future studies using the Ba/F3 transformation assay verify the complete sequence of the ectopically expressed transgene prior to pathway characterization and publication of results.…”

Section: Introductioncontrasting

confidence: 99%

“…A previous study investigating another in vitro transforming CSF2RB mutation found that the transformation rate of infected cells increased with the time in culture after viral infection and before factor withdrawal [32]. In our study, we observed no consistent effect of culture time on transformation rate with any of the mutations tested (Figure 3A).…”

Section: Resultscontrasting

confidence: 74%

“…Distinguishing between the passenger mutations and the functionally active acquired mutations would require laborious empirical investigation, detracting from the scalability of this model system and thus making it an impractical solution. We have not attempted to determine the functionality of observed acquired mutations, though specific extracellular deletions have been previously shown to result in receptor activation of CSF2RB [32, 43]. …”

Section: Discussionmentioning

confidence: 99%

“…It is possible that weak mutations are barely capable of transforming cells to become factor independent, possibly requiring a high level of expression, and the majority of cells fail to reach that state. It is also possible that some mutations need a second, cooperating mutation as proposed by earlier studies [32]. …”

Section: Discussionmentioning

confidence: 99%

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Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations

et al. 2017

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The identification and functional validation of potentially oncogenic mutations in leukemia is an essential step toward a future of personalized targeted therapy. To assess the oncogenic capacity of individual mutations, reliable and scalable in vitro experimental approaches are required. Since 1988, researchers have used the IL-3 dependent Ba/F3 transformation assay to validate the oncogenic potential of mutations to drive factor-independent growth. Here we report a previously unrecognized phenomenon whereby Ba/F3 cells, engineered to express weakly transforming mutations, present with additional acquired mutations in the expressed transgene following factor withdrawal. Using four mutations with known transformative capacity in three cytokine receptors (CSF2RB, CSF3R and IL7R), we demonstrate that the mutated receptors are highly susceptible to acquiring additional mutations. These acquired mutations of unknown functional significance are selected by factor withdrawal but appear to exist prior to the removal of growth factor. This anomaly has the potential to confound efforts to both validate and characterize oncogenic mutations in leukemia, particularly when it is not standard practice to sequence validate cDNAs from transformed Ba/F3 lines. We present specific recommendations to detect and mitigate this phenomenon in future research using Ba/F3 transformation assays, along with methods to make the Ba/F3 assay more quantitative.

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Section: Introductioncontrasting

confidence: 99%

Section: Resultscontrasting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations

et al. 2017

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“…FMEV-YFP is similar to FMEV-GFP, except the eGFP coding sequence is replaced with that of the enhanced yellow fluorescent protein (eYFP; BD Biosciences, Heidelberg, Germany). To generate a Cre-recombinase expression vector (FMEV-CRE), a cassette containing the simian virus (SV)-40 promoter and the Cre coding region 21 was placed downstream of the eGFP gene in a FMEV-vector. Retroviral pseudotypes were prepared as previously described 14 with either the envelope (Env) protein of the Moloney murine leukemia virus 22 (for infection of mouse cells) or the feline endogenous retrovirus RD114 23 (for infection of human cells).…”

Section: Retroviral Vectors and Generation Of Infectious Pseudotyped mentioning

confidence: 99%

A dominant-negative mutant of C/EBPα, associated with acute myeloid leukemias, inhibits differentiation of myeloid and erythroid progenitors of man but not mouse

Schwieger

Löhler

Fischer

et al. 2004

Blood

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The CCAAT/enhancer binding protein alpha (C/EBP␣) is an essential transcription factor for granulocytic differentiation. C/EBP␣ mutations are found in approximately 8% of acute myeloid leukemia (AML) patients. Most of these mutations occur in the N-terminal coding region, resulting in a frame shift and the enhanced translation of a dominant-negative 30-kDa protein, which may be responsible for the differentiation block observed in AML. To test this hypothesis, we introduced a cDNA encoding an Nterminal mutated C/EBP␣ (mut10) into primary hematopoietic progenitors using a retroviral vector. Expression of mut10 in human CD34 ؉ cord blood cells dramatically inhibited differentiation of both myeloid and erythroid lineages. Immunohistochemical analysis demonstrated coexpression of both myeloid and erythroid markers in the immature transformed cells. Surprisingly, mut10 did not block myelocytic differentiation in murine progenitors but did alter their differentiation kinetics and clonogenicity. Experiments were performed to confirm that the differential effect of mut10 on murine and human progenitors was not due to species-specific differences in C/EBP␣ protein sequences, expression levels, or inefficient targeting of relevant cells. Taken together, our results underline the intrinsic differences between hematopoietic controls in mouse and human and support the hypothesis that mutations in CEBPA are critical events in the disruption of myeloid differentiation in AMLs. IntroductionThe hallmark of acute leukemia is the clonal, neoplastic proliferation of immature hematopoietic cells. The observed inhibition or complete arrest in differentiation can conceivably be due to an indirect mechanism, in which genetic mutations that disrupt cell cycle controls support proliferation at the expense of differentiation, and/or due to genetic abnormalities that directly interfere with the differentiation program. In support of the latter hypothesis, growing evidence suggests that the transcription factors that play pivotal roles in lineage-specific differentiation may be important targets of transformation in acute leukemias. 1 The basic zipper (B-ZIP) transcription factor CCAAT/enhancer binding protein ␣ (C/EBP␣) and the ETS-family transcription factor PU.1 are 2 major regulators of myeloid differentiation. 2,3 Unlike PU.1, which governs transcription of a wide spectrum of myeloid-specific genes found in both early hematopoietic stem cells and mature monocytes and granulocytes, C/EBP␣ regulation has a more specific function in granulopoiesis. Indeed, C/EBP␣ knockout mice show a selective block in the differentiation of granulocytes, including neutrophils and eosinophils. 4 Furthermore, the constitutive expression of C/EBP␣ in myeloid progenitors results in the induction of granulocytic development and partial inhibition of monocytic and erythroid development. [5][6][7] Several independent studies have demonstrated mutations in the CEBPA gene encoding C/EBP␣ in approximately 8% of acute myeloid leukemia (AML). [8][9][10][11] These m...

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Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose

Schwieger

Lange

et al. 2003

Experimental Hematology

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Functional identification of secondary mutations inducing autonomous growth in synergy with a truncated interleukin-3 receptor

Cited by 6 publications

References 51 publications

Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations

Analysis of acquired mutations in transgenes arising in Ba/F3 transformation assays: findings and recommendations

A dominant-negative mutant of C/EBPα, associated with acute myeloid leukemias, inhibits differentiation of myeloid and erythroid progenitors of man but not mouse

Predictable and efficient retroviral gene transfer into murine bone marrow repopulating cells using a defined vector dose

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