Clinical relevance of recurrent allele-specific recombination expressing the WNT10Bivs1 allele variant in acute myeloid leukemia

Background: The WNT/β-catenin pathway play a critical function in the regulation of cell proliferation, differentiation, and apoptosis, playing a major role in fueling stem cell activity and sustaining tissue regeneration in a dose dependent manner in several adult stem cell niches including bone marrow. Previous results obtained by our research team provided direct evidences that WNT/β-catenin signaling is diffusely activated in the AC133+acute myeloid leukemia (AML) cells. The mRNA in situ detection analysis revealed that WNT10B results to be expressed in leukemic blasts as well as in stromal-like cells, suggesting an autocrine/paracrine mechanism of Wnt signaling induction in the leukemic bone marrow microenvironment. Conversely, the activation of Wnt signaling, marked by expression of the dephosphorylated β-catenins, is restricted only to a smaller subpopulation of AC133bright cells. Aims: In the present study, focusing our attention on the major locus associated in hematopoiesis to the regenerative function, we provide evidences for a recurrent rearrangement involving the WNT10B locus within intron 1 (IVS1). Moreover, we demonstrated the consequent expression of a non-physiological transcript variant, WNT10BIVS1, retaining 77 nucleotide of IVS1 and lacking exon1, and we analyzed the significance in AML. Methods: In order to provide accurate quantification of mRNA levels of WNT10B and the related WNT10BIVS1 transcript variant and analyze the clinical relevance of their expression, we carried out the gene expression analysis by Droplet DigitalTM PCR on mononucleated cells derived from n=125 AML patients [de novo, N=118 (intermediate-adverse risk N=70; favorable risk N=48); therapy-related, N=7, informed consent was obtained]. Analyzing patients according to the three main scoring systems, formerly the Medical Research Council (MRC), European LeukemiaNet (ELN), and National Comprehensive Cancer Network (NCCN), we were able to distinguish groups of patients at different outcomes with a statistically significant wise. Results: We observed that canonical WNT10B mRNA was highly expressed in all de novo AML patients here examined, while WNT10BIVS1 mRNA transcript levels resulted undetectable in patients classified with favorable-risk (p <0.001). Furthermore, we demonstrated absence of both WNT10B and WNT10BIVS1 expression in therapy-related patients (p <0.005). Moreover, we have also evaluated the effects of transient wnt10b overexpression on early hematopoiesis in the zebrafish model and therefore, to this aim wnt10b synthetic mRNA was microinjected in the zebrafish zygote to mimic, in vivo, the Wnt signaling overexpression we had previously observed in AC133bright AML cells. Interestingly, an increase of erythromyeloid progenitors, and a simultaneous reduction in the number of circulating neutrophils is detected. Summary/Conclusions: The results presented here provide compelling evidence that regeneration-associated Wnt signaling exceeds the homeostatic range in the majority of human AML cases. Taking these issues into consideration, we revealed that it is possible to recognize three distinct WNT10B/ WNT10BIVS1 patterns, suggesting a potential role of WNT10BIVS1 transcript as a marker for the intermediate-adverse risk AML patients. These findings, if confirmed in a larger population of patients, may help to refine diagnostic or prognostic criteria for previously described neoplasms, and to introduce newly recognized disease entities possibly characterized by distinct causative pathogenic mechanisms.