Unraveling functional consequences of AID off-target mutations in CLL

SEIJA NOÉ; YAN XIAO-YEN; RITA URIA; CHIORAZZI NICHOLAS; AMPARO RICO; SEPULVEDA-YANEZ, JULIETA HAYDEE; PALACIOS FLORENCIA; GIMENA DOS SANTOS; NAVARRETE, MARCELO ALEJANDRO; JORGE PUELMA; PABLO MORANDE; MARQUEZ, MARIA ELENA; LANDONI ANA INÉS; OPPEZZO PABLO

virtual

Workshop; iwCLL 2021; 2021

iwCLL comite

Neoplasms become more aggressive by the outgrowth of malignant subclones harboring additional DNA mutations. Activation-induced cytidine deaminase (AID), an enzyme that allows normal B lymphocytes to develop more effective immunoglobulin (Ig)-mediated immunity, may also mutate non-Ig genes involved in oncogenesis and tumor evolution. In chronic lymphocytic leukemia (CLL), AID expression correlates with poor prognosis, suggesting its role in disease progression. To understand the mechanistic basis of this correlation, we developed two transgenic models overexpressing AID in the murine model of CLL (Eµ/TCL1). Both models hereafter called DT-TCL1/AID develop more aggressive leukemias with shorter survivals, linking constitutive AID expression in the leukemic clone with enhanced cell proliferation. Whole exome sequencing (WES) of DT-AID leukemic clones and their proliferating fractions revealed novel AID-induced mutations in genes involved in CLL progression and demonstrated that AID can induce mutations that were previously described in human cancer driver genes. Notably, some of these changes occurred at homologous positions, leading to identical or chemically-similar amino acid substitutions as in the human disease, suggesting these are driver mutations selected for their oncogenic effects (Morande and Yan et al, Blood. 2021, Mar 2). Here, we focus on the consequences of AID mutations on selected off-target genes to further explore the role of AID expression in human CLL progression. To this aim, we performed WES on leukemic clones from 12 CLL patients with progressive disease and systematically analyzed genes affected by AID mutations in these patients and concomitantly in our murine models. Patients were selected based on continuous AID expression (positive AID mRNA in peripheral blood -PB-, evaluated every 3 months until treatment), unmutated IgVH profile, Binet Stage B or C, and short time to first treatment (< to 2 years). WES data from this CLL cohort were analyzed for mutational context, focusing on putative AID hotspots (c-AID; nc-AID, and NCG). Our results show that progressive patients expressing AID in PB reveal a mutational pattern dominated by a c-AID signature, which is different from that occurring in patients with indolent disease. This is consistent with our data regarding the signature profile of the DT-AID mice models recently published (Morande and Yan et al, Blood. 2021). Interestingly, we also observed that preferentially targeted 3’ untranslated regions (UTRs) over 5’ UTRs; this was also found in the DT-AID mice. Due the importance of 3’ UTRs in regulating gene expression, often mediated by microRNAs, we are validating in a distinct CLL cohort these specific AID off-target mutations. To this, 10 specific genes - CD38, ROR1, ROS1, PRKCH, IL17A, IL17RA, AKAP13, TMEM176A, IKZF1 and PAX5 - have been selected based on the percentage of the mutation frequency within the clone and their putative role in the leukemic progression, and differential expression, at mRNA and proteins level are being evaluated with the goal to provide additional evidence that links off-target AID activity with tumor progression in CLL.