PS1454 CAG PROMOTER DRIVEN C‐MYC OVEREXPRESSION CAUSES RAPIDLY LETHAL CARDIOMYOPATHY
Authors: Kovacs S. Bojtine , Basu T. , Pahl H.L. 
Published: June 2019

Background:

We have previously reported that mice expressing elevated levels of NFE2 display a myeloproliferative phenotype which spontaneously transforms to acute leukemia. Upon leukemic transformation, a high percentage of mice acquire duplications or amplification of the murine c‐myc locus. The CMV early enhancer/chicken‐beta‐actin (CAG) promoter has been reported to direct high level heterologous gene expression specifically to hematopoietic stem cells and myeloid progenitors.

Aims:

We hypothesized that elevated expression of both NFE2 and c‐myc synergize to induce leukemic transformation. Therefore, we compared the hematological phenotype of mice carrying either augmented NFE2 or c‐myc levels alone to mice overexpressing both genes.

Methods:

We crossed mice expressing a CAG‐driven tetracycline activator (CAG‐rtTA3) with the tet‐O‐myc mouse strain, in order to achieve high levels of doxycycline‐inducible c‐myc expression in the hematopoietic compartment. Double transgenic CAG‐myc mice were subsequently bred to heterozygous knock‐in mice expressing the NFE2‐D297–300 mutation of the endogenous promoter, generating both CAG‐myc‐NFE2‐wt and CAG‐myc‐NFE2mut mice. C‐myc expression was induced by doxycycline treatment administered through various routes, including drinking water, oral gavage and food pellets.

Results:

Unexpectedly both CAG‐myc and CAG‐myc‐NFE2 mice died rapidly, between 6 and 7 days following c‐myc induction, independent of the route of doxycycline administration. Autopsy and histological analysis revealed marked cardiomyocyte hypertrophy, with heart volume more than double the wt control (Fig. 1). In addition to hematopoietic stem and progenitor cells, the CAG promoter has been reported to direct expression to cardiomyocytes. C‐myc overexpression in adult cardiomyocytes by tet‐mediated c‐myc induction has previously been shown to cause cell cycle re‐entry that leads to cardiomyopathy and hypertrophy. However, it was not expected that the CAG promoter, seen as a “highly effective tool for probing hematopoietic development and disease”, would have a similarly cardiotoxic effect.

Fig. 1: Pathology and histology of hearts following doxycycline induction of CAG‐myc expression. (A) representative autopsy finding from a wt control mouse (left) and a CAG‐myc‐NFE2‐wt mouse. Results are representative of n = 16 CAG‐myc mice. (B) Hematoxylin Eosin (H&E) staining of histological sections from CAG‐myc‐NFE2 wt and CAG‐myc‐NFE2mut mice before and after doxycycline induction. 200x magnification, size bars represent 50 mm.

Summary/Conclusion:

While our murine model did not allow us to investigate our hypothesized synergistic effect between c‐myc and NFE2 in leukemic transformation, the CAG‐myc mouse strain generated constitutes an excellent model for studying rapidly lethal cardiomyopathy. Moreover, our data serve to alert researchers in the hematopoietic field to possible side effects of using the CAG promoter to drive gene expression in hematopoietic stem and progenitor cells.

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