C22orf2 Gene (22q13.1) Translocation Downstream of Bcr Breakpoint (22q11) to the Derivative 9 Chromosome Is Associated with Reduced Expression of Its Protein, the Beta-Catenin Antagonist Chibby, in Chronic Myeloid Leukemia Progenitors
Authors: Aluigi Michela , Mancini Manuela , Leo Elisa , Castagnetti Fausto , Baldazzi Carmen , Barbieri Enza , Santucci Maria Alessandra 
Published: October 2019

Abstract
Abstract 1676
Chibby is a 14.5 kDa proteins encoded by C22orf2 gene located at chromosome 22q13-1. Its major function is to antagonize beta catenin by competing for binding with Tcf/Lef transcription factors and interacting with 14-3-3 scaffolding proteins thereby promoting cytoplasmatic compartmentalization into a stable tripartite complex with beta catenin. Chibby relative vicinity to the Bcr breakpoint on chromosome 22 and its antagonistic role on beta catenin, a critical signal for self renewal and growth of CML stem cells addressed us to investigate Chibby expression in chronic myeloid leukemia (CML) and its impact on the disease prognosis. We first compared for Chibby protein expression levels in mononuclear cell fractions (MNCs) from bone marrow samples of CML patients (n. 28) at clinical diagnosis (n. 18) or in more advanced stages of the disease (n. 10) and peripheral blood aphaeresis of normal persons. A significant reduction of Chibby was detected in 14 out of 28 CML samples analyzed (p<0.01 or less) independent from the disease stage. Semi-quantitative and real-time PCR analysis revealed a significant reduction of Chibby transcript in 6/15 samples. We then investigated the expression of Chibby in the putative stem cell compartment (CD34+) isolated from bone marrow MNCs of CML patients (n.3) at diagnosis. Both Chibby protein and transcript were significantly lower (p<0.001 or less) in CD34+ cell fraction compared to the whole MNC, supporting that the down-regulation of Chibby may have a prominent role in the constitutive activation of beta catenin in an early CML progenitor compartment. Further investigation revealed that Chibby expression levels were raised in MNCs from CML patients who achieved a major molecular response under IM therapy compared with the levels seen at diagnosis. The findings support that Chibby down-regulation is indeed associated with t(9;22) translocation and is restricted to the leukemic clone. Finally, we performed FISH analysis on MNCs of all CML patients by means of 2 different probes mapping the entire region on chromosome 22q13.1 where Chibby gene is located and found that one Chibby allele is translocated to the derivative 9 chromosome still fused to upstream Bcr sequences.
In conclusion, results presented here let distinguish the translocation of C22orf2 gene from chromosome 22q13-1 to the derivative chromosome 9 as a genetic lesion associated with Bcr breakpoint at 22q11 of CML. Such a genetic lesion is almost invariably associated with the down-regulation of Chibby which is likely involved in the constitutive activation of beta catenin driving the proliferative advantage of leukemic stem cells over their normal counterpart. Further investigation is required to elucidate whether Chibby down-regulation in CML is driven by transcriptional mechanisms (such as the impairment of gene regulatory sequences) or by post-transcriptional events (eventually affecting the protein interactions and stability).

Disclosures:
Castagnetti: Novartis: Honoraria; Bristol Myers Squibb: Honoraria.