Page 217 - 2020_02-Haematologica-web

BIRC3 mutations in CLL
through MAP3K14 stabilization as the predicted function- al consequence of BIRC3 mutations in CLL.
The non-canonical NF-κB signaling was profiled by immunoblotting in B-cell tumor cell lines and primary CLL cells with different genetic make-up in the non-canonical NF-κB pathway to verify whether BIRC3 mutations lead to constitutive non-canonical NF-κB activation. Additional genetic features of the above-mentioned cell lines and pri- mary CLL cells are shown in Online Supplementary Table S3. In the VL51 splenic marginal zone lymphoma cell line and in the MEC1 CLL cell lines, both harboring endoge- nous truncating mutations of the BIRC3 gene, non-canon- ical NF-κB signaling was constitutively active, as docu- mented by the stabilization of MAP3K14, phosphoryla- tion of NF-κB2, its processing from p100 to p52, as well as the nuclear localization of p52 (Figure 1B-D). Consistent with the biochemical clues of non-canonical NF-κB activa- tion, the gene expression signature of the VL51 and MEC1 cell lines was significantly enriched in non-canonical NF- κB target genes (Figure 1E, F). Non-canonical NF-κB signal- ing in BIRC3-mutated cells was consistent with that in mantle cell lymphoma cell lines known to harbor a dis- rupted TRAF3/MAP3K14-TRAF2/BIRC3 negative regula- tory complex by loss of TRAF3 or TRAF2.14 Like BIRC3- mutated cell lines, primary CLL samples harboring inacti- vating mutations of BIRC3 also showed stabilization of MAP3K14 and NF-κB2 processing from p100 to p52 (Figure 1C), thus confirming that non-canonical NF-κB activation is also a feature of primary cells harboring BIRC3 variants. MAP3K14 stabilization is largely associated with BIRC3 mutations. Indeed all seven cases harboring non-canonical NF-κB genetic lesions showed either a strong or a slight MAP3K14 band, while, conversely, only one out of five cases lacking a non-canonical NF-κB lesion had MAP3K14 expression (Fisher exact test, P=0.01).
MAP3K14 was genetically targeted by shRNA to test whether BIRC3-mutated cells are addicted to its stabiliza- tion. Compared to non-targeting shRNA, the most effi- cient anti-MAP3K14 shRNA-D resulted in a partial silenc- ing of MAP3K14 and in decreased NF-κB2 processing from p100 to p52. This translated into a reduced cell viability of
AB
the BIRC3-mutated VL51 cell line transduced with shRNA-D. This observation indicates that MAP3K14 sta- bilization is a vulnerability of BIRC3-mutated cells (Figure 2). In order to test the contribution of BTK to non-canon- ical NF-κB signaling when it is activated through BIRC3 mutations, BIRC3-mutated cell lines, as well as cell lines harboring a disrupted or competent TRAF3/MAP3K14- TRAF2/BIRC3 negative regulatory complex were treated with ibrutinib at different dosages and non-canonical NF- κB signaling activation was probed by immunoblotting of the NF-κB2 processing from p100 to p52. Processing from p100 to p52 was unaffected by ibrutinib treatment in cell lines harboring BIRC3 mutations (Figure 3) or a disrupted TRAF3/MAP3K14-TRAF2/BIRC3 negative regulatory complex, consistent with the notion that BIRC3 mutations activate non-canonical NF-κB by bypassing BTK blockade by ibrutinib.14
BIRC3 mutations confer resistance to fludarabine in primary chronic lymphocytic leukemia cells
We performed in vitro pharmacological studies on pri- mary CLL cells to verify the vulnerabilities of BIRC3- mutated cells. CLL cells purified from patients carrying BIRC3 mutations were treated with increasing doses of fludarabine. Drug-induced apoptosis was compared to that of samples harboring TP53 mutations, which repre- sent a control for fludarabine resistance. CLL cells devoid of genetic lesions in either BIRC3 or TP53 were used as a control for fludarabine sensitivity. The molecular charac- teristic of the ex-vivo CLL cells are listed in Online Supplementary Table S4.
BIRC3-mutated cells showed delayed fludarabine- induced cell death, as no response was observed after 24 h of treatment, at variance with TP53- and BIRC3-wildtype samples. At this time point, cell viability curves of BIRC3- mutated samples overlapped almost completely with those of TP53-disrupted samples, which are known to be fludarabine resistant (Figure 4A). At 48 h, the viability of BIRC3-mutated cells was lower than that of TP53-mutat- ed samples, but higher than that of TP53- and BIRC3-wild- type samples (Figure 4B).
Figure 2. Knockdown of MAP3K14 by RNA interference in VL51 cells. (A) Western blot analysis for MAP3K14 expression and for NF-κB2 processing of p100 to p52. (B) VL51 cell viability assessed by trypan blue after transduction with lentiviral vectors expressing the shRNA-D_MAP3K14 (shD: in red), a scrambled shRNA (scram- bled: in blue), and in non-transfected cells (CTRL: in green).
haematologica | 2020; 105(2)
451