Biallelic Deletion 13q in Chronic Lymphocytic Leukemia Treated with Targeted Agents

Monoallelic deletion 13q (del(13q)) is the most common genomic aberration in chronic lymphocytic leukemia (CLL) and is associated with favorable prognosis when occurring as sole abnormality. In our analysis of genetic markers and outcome in CLL2-BAG, BCG, BIG, and BIO trials (CLL2-BXX, n= 242) of the German CLL study group, we identified among other genetic factors, the variant fraction of biallelic del(13q) (bidel(13q)) to be increased at disease relapse compared to baseline (median: 67% vs. 12.5%; n=7/42). This increase was significant with venetoclax based treatments (5/16; Yosifov et al., EHA 2023). Here, we further characterized associations of bidel(13q) with other genetic characteristics in the pretreatment setting and at relapse. Moreover, we functionally studied the role of bidel(13q) in response to venetoclax in ex vivo primary CLL samples and using in vitro models.

Firstly, we analyzed associations of bidel(13q) in n=3,327 treatment naive CLL. At baseline, bidel(13q) was significantly associated with lower incidence of the high and intermediate risk factors, unmutated IGHV (uIGHV), del(11q), trisomy 12 (all p<0.001) and del(17p) (p=0.015). On the contrary, when analyzing 55 relapse samples from the CLL2-BXX trials including BAAG and GIVe, bidel(13q) always co-occurred with del(17p). In 7 of 9 samples with increasing bidel(13q), the proportion of cells with del(17p) also increased, while in 2 of 9 samples bidel(13q) and del(17p) showed opposing trends. Next, we aimed to study the biological relationship between bidel(13q) and response to venetoclax.

MicroRNAs (miR) 15a and 16-1 present in the minimally deleted region of del(13q) in CLL were reported to regulate the anti-apoptotic proteins BCL2 and MCL1. To study the impact of biallelic loss of the miRs on venetoclax response, we screened CLL and mantle cell lymphoma (MCL) cell lines for a suitable model with sensitivity to venetoclax and presence of at least one wildtype 13q allele. MCL cell lines MAVER-1 (IC¬50 67nM) and MINO-1 (IC50 12nM) showed sensitivity to venetoclax in vitro. Fluorescence in situ hybridization identified MAVER-1 cells to harbor an intact 13q14 chromosomal locus, hence it was selected for further studies. The miRs 15a and 16-1 were knocked out in MAVER-1 cells by CRISPR-Cas9 and the knockout efficiency was verified at the DNA and RNA levels using PCR and qPCR, respectively.

Western blotting for anti and proapoptotic protein expression did not show substantial difference between the knockout cells and controls. Alternative mechanisms mediating a strong BCL2 upregulation could potentially mask the impact of miR knockout in MAVER-1 cells. However, cell cycle progression to S phase in the knockout cell lines was marginally increased (28% vs 20%), and the differences were not statistically significant. Analysis of venetoclax response and apoptotic priming using BH3 profiling identified no significant difference between the scrambled controls and miR knockouts. Since interaction with CD40L or TLR agonist CpG is known to alter expressions and dependencies on antiapoptotic proteins, we exposed the cells to CD40L and CpG prior to performing BH3 profiling. The miR knockout cells showed a decrease in apoptotic priming and an inferior response to venetoclax when exposed to CpG, however, the differences were not statistically significant (p=0.13).

We further characterized responses to venetoclax in the presence and absence of stimulation with CD40L, CpG, IL-2, IL-15, and IL-21 in samples with bidel(13q) and matched controls (n=6 each). All samples showed diminished responses to venetoclax upon stimulation with co-factors but samples with bidel(13q) and uIGHV showed a greater increase in venetoclax IC50 upon stimulation compared to uIGHV control samples (median: 26.1 vs. 6.3-fold). BH3 profiling identified decreased apoptotic priming in samples with bidel(13q) and uIGHV compared to corresponding uIGHV controls (median Δ priming to 0.1µM BIM: 16% vs 61%).

In summary, though bidel(13q) is strongly associated with good prognostic genetic factors and favorable outcome when occurring alone, the percentage of cells with bidel(13q) was increased at relapse after therapy with targeted agents, especially venetoclax in the CLL2-BXX trials and was associated with del(17p). Ex vivo analyses hint at diminished apoptotic priming when bidel(13q) occurs in the background of poor risk factors such as unmutated IGHV.