Endogenous tumor suppressor microRNA-193b: Therapeutic and prognostic value in acute myeloid leukemia

Purpose Dysregulated microRNAs are implicated in the pathogenesis and aggressiveness of acute myeloid leukemia (AML). We describe the effect of the hematopoietic stem-cell self-renewal regulating miR-193b on progression and prognosis of AML. Methods We profiled miR-193b-5p/3p expression in cytogenetically and clinically characterized de novo pediatric AML (n = 161) via quantitative real-time polymerase chain reaction and validated our findings in an independent cohort of 187 adult patients. We investigated the tumor suppressive function of miR-193b in human AML blasts, patient-derived xenografts, and miR-193b knockout mice in vitro and in vivo. Results miR-193b exerted important, endogenous, tumor-suppressive functions on the hematopoietic system. miR-193b-3p was downregulated in several cytogenetically defined subgroups of pediatric and adult AML, and low expression served as an independent indicator for poor prognosis in pediatric AML (risk ratio 6 standard error, 20.56 6 0.23; P = .016). miR-193b-3p expression improved the prognostic value of the European LeukemiaNet risk-group stratification or a 17-gene leukemic stemness score. In knockout mice, loss of miR-193b cooperated with Hoxa9/Meis1 during leukemogenesis, whereas restoring miR-193b expression impaired leukemic engraftment. Similarly, expression of miR-193b in AML blasts from patients diminished leukemic growth in vitro and in mouse xenografts. Mechanistically, miR-193b induced apoptosis and a G1/S-phase block in various human AML subgroups by targeting multiple factors of the KIT-RAS-RAF-MEK-ERK (MAPK) signaling cascade and the downstream cell cycle regulator CCND1. Conclusion The tumor-suppressive function is independent of patient age or genetics; therefore, restoring miR-193b would assure high antileukemic efficacy by blocking the entire MAPK signaling cascade while preventing the emergence of resistance mechanisms

The pathologic effect of a novel neomorphic Fgf9(Y162C) allele is restricted to decreased vision and retarded lens growth.

Fibroblast growth factor (Fgf) signalling plays a crucial role in many developmental processes. Among the Fgf pathway ligands, Fgf9 (UniProt: P54130) has been demonstrated to participate in maturation of various organs and tissues including skeleton, testes, lung, heart, and eye. Here we establish a novel Fgf9 allele, discovered in a dominant N-ethyl-N-nitrosourea (ENU) screen for eye-size abnormalities using the optical low coherence interferometry technique. The underlying mouse mutant line Aca12 was originally identified because of its significantly reduced lens thickness. Linkage studies located Aca12 to chromosome 14 within a 3.6 Mb spanning interval containing the positional candidate genes Fgf9 (MGI: 104723), Gja3 (MGI: 95714), and Ift88 (MGI: 98715). While no sequence differences were found in Gja3 and Ift88, we identified an A→G missense mutation at cDNA position 770 of the Fgf9 gene leading to an Y162C amino acid exchange. In contrast to previously described Fgf9 mutants, Fgf9(Y162C) carriers were fully viable and did not reveal reduced body-size, male-to-female sexual reversal or skeletal malformations. The histological analysis of the retina as well as its basic functional characterization by electroretinography (ERG) did not show any abnormality. However, the analysis of head-tracking response of the Fgf9(Y162C) mutants in a virtual drum indicated a gene-dosage dependent vision loss of almost 50%. The smaller lenses in Fgf9(Y162C) suggested a role of Fgf9 during lens development. Histological investigations showed that lens growth retardation starts during embryogenesis and continues after birth. Young Fgf9(Y162C) lenses remained transparent but developed age-related cataracts. Taken together, Fgf9(Y162C) is a novel neomorphic allele that initiates microphakia and reduced vision without effects on organs and tissues outside the eye. Our data point to a role of Fgf9 signalling in primary and secondary lens fiber cell growth. The results underline the importance of allelic series to fully understand multiple functions of a gene

Regular equatorial lens histology and fiber cell size of <i>Fgf9^Y162C</i> mutants.

<p>(A,B) Morphology of (A) four-month-old C57BL/6J control (WT) and (B) homozygous <i>Fgf9^Y162C</i> (A/A) lenses is shown. The mutants display a regularly developed lens bow. L, lens; R, retina. (C) Mean lens fiber cell thickness at the age of four months calculated by averaging the breadth of ten fiber cell layers in the equatorial outer cortex. Lens fiber cell size did not differ significantly in the homozygous <i>Fgf9^Y162C</i> mutants. Values are means ± standard deviation (<i>n</i> = 5).</p

Haplotype analysis of the <i>Aca12</i> mutation and sequence analysis of the <i>Fgf9</i> coding region.

<p>(A) The <i>Aca12</i> mutation is localized between the flanking microsatellite markers <i>D14Mit268</i> and <i>D14Mit234</i>. Black boxes illustrate the presence of two C3H marker alleles (recombination between microsatellite marker and <i>Aca12</i>); white boxes illustrate heterozygosity of the markers for the C3H and C57BL/6J allele (lack of recombination). The number of G3 progeny carrying the particular recombination pattern is given below the boxes. The total number of recombination (R) events between neighbouring markers is shown to the right of the boxes, including the calculated relative genetic distances (cM ± standard deviations). In total, we tested 175 G3 animals. (B) Sequence analysis of genomic DNA of a heterozygous mutant shows an A→G transition (black arrow) at cDNA position 855 of the <i>Fgf9</i> gene resulting in a Y162C amino acid exchange in the Fgf9 protein.</p

Reduced visual acuity in the <i>Fgf9^Y162C</i> mutants.

<p>Spatial frequency thresholds indicated a reduced vision in the <i>Fgf9^Y162C</i> mutants. Values represent means ± standard deviation of measurements from twelve C57BL/6J controls (WT), ten heterozygous <i>Fgf9^Y162C</i> mutants (A/+), and ten homozygous <i>Fgf9^Y162C</i> carriers (A/A). *<i>p</i><0.001.</p

<i>Fgf9^Y162C</i> mutants exhibit regular sexual development.

<p>Sex genotyping PCR amplified a <i>Sry</i> fragment (266 bp) indicating the presence of a Y chromosome in all tested male samples, but in none of the analyzed female probes. WT, C57BL/6J control (<i>n</i> = 5); A/+, heterozygous <i>Fgf9^Y162C</i> mice (<i>n</i> = 6–9); A/A, homozygous <i>Fgf9^Y162C</i> mice (<i>n</i> = 6–12); M, marker.</p

Decreased <i>Fgf9^Y162C</i> lens thickness at postnatal stages and retarded postnatal lens growth in <i>Fgf9^Y162C</i> mutants.

<p>(A,B) Mean lens thickness (polar diameter) of homozygous <i>Fgf9^Y162C</i> mutants (dashed lines) obtained by OLCI at different postnatal stages are compared to the C57BL/6J control data (solid lines). Mutants exhibit significantly reduced lens sizes at all tested stages. (A) females; (B) males. (C,D) Postnatal lens growth of homozygous <i>Fgf9^Y162C</i> mice (white bars) and C57BL/6J controls (black bars) is tracked by OLCI. In the mutants, lens growth is most strikingly reduced at early postnatal stages. (C) females; (D) males. Values are means ± standard deviation (<i>n</i> = 10–12). *<i>p</i><0.01; **<i>p</i><0.001.</p

<i>Fgf9^Y162C</i> mice exhibit regular functionality of the retina.

<p>Electroretinograms of six-month-old C57BL/6J control (WT) and homozygous <i>Fgf9^Y162</i>^C (A/A) mice are shown. The mutants exhibit a regular electrical response to the given light flashes. (A), a-wave amplitude; (B), b-wave amplitude.</p