PURPOSE: In previous analyses of the MURANO study, fixed-duration venetoclax plus rituximab (VenR) resulted in improved progression-free survival (PFS) compared with bendamustine plus rituximab (BR) in patients with relapsed or refractory chronic lymphocytic leukemia (CLL). At the 4-year follow-up, we report long-term outcomes, response to subsequent therapies, and the predictive value of molecular and genetic characteristics. PATIENTS AND METHODS: Patients with CLL were randomly assigned to 2 years of venetoclax (VenR for the first six cycles) or six cycles of BR. PFS, overall survival (OS), peripheral-blood minimal residual disease (MRD) status, genomic complexity (GC), and gene mutations were assessed. RESULTS: Of 389 patients, 194 were assigned to VenR and 195 to BR. Four-year PFS and OS rates were higher with VenR than BR, at 57.3% and 4.6% (hazard ratio [HR], 0.19; 95% CI, 0.14 to 0.25), and 85.3% and 66.8% (HR, 0.41; 95% CI, 0.26 to 0.65), respectively. Undetectable MRD (uMRD) at end of combination therapy (EOCT) was associated with superior PFS compared with low MRD positivity (HR, 0.50) and high MRD positivity (HR, 0.15). Patients in the VenR arm who received ibrutinib as their first therapy after progression (n = 12) had a reported response rate of 100% (10 of 10 evaluable patients); patients subsequently treated with a venetoclax-based regimen (n = 14) had a reported response rate of 55% (six of 11 evaluable patients). With VenR, the uMRD rate at end of treatment (EOT) was lower in patients with GC than in those without GC ( P = .042); higher GC was associated with shorter PFS. Higher MRD positivity rates were seen with BIRC3 and BRAF mutations at EOCT and with TP53, NOTCH1, XPO1, and BRAF mutations at EOT. CONCLUSION: Efficacy benefits with fixed-duration VenR are sustained and particularly durable in patients who achieve uMRD. Salvage therapy with ibrutinib after VenR achieved high response rates. Genetic mutations and GC affected MRD rates and PFS.

The current interest in expression of groups of functionally related genes creates a demand for novel experimental tools. We describe a multiplex ligation‐dependent amplification procedure (RT‐MLPA), which accurately quantifies up to 45 transcripts of interest in a one‐tube assay. The output, a set of fluorescent DNA fragments, is analysed via capillary sequencer and spreadsheet software. The procedure is highly sensitive and reproducible over a 100‐fold range of input RNA, with excellent compatibility with RT–PCR and microarrays. We targeted two comprehensive sets of human genes: 35 apoptosis regulators and 30 genes involved in inflammation. Both probe sets accurately assessed specific changes in gene expression in two relevant model systems. Stimulation of lymphocytes with various Toll‐like receptor (TLR) ligands induced distinct inflammatory profiles. Furthermore, osteosarcoma cells treated with cytostatic drugs showed as primary response strong up‐regulation of the apoptogenic p53‐inducible PUMA transcript. Suppression by RNAi validated that indeed Puma expression was responsible for apoptosis induction. Thus, RT‐MLPA enables relevant changes in transcription patterns to be quickly pinpointed and guide further experiments. This can be an advantage compared to hypothesis‐free whole genome screens where large numbers of differentially expressed genes can obscure functional interpretation.

Chronic lymphocytic leukemia (CLL) cells cycle between lymph node (LN) and peripheral blood (PB) and display major shifts in Bcl-2 family members between those compartments. Specifically, Bcl-XL and Mcl-1, which are not targeted by the Bcl-2 inhibitor venetoclax, are increased in the LN. Since ibrutinib forces CLL cells out of the LN, we hypothesized that ibrutinib may thereby affect expression of Bcl-XL and Mcl-1 and sensitize CLL cells to venetoclax. We investigated expression of Bcl-2 family members in patients under ibrutinib or venetoclax treatment combined with dissecting functional interactions of Bcl-2 family members in an in vitro model for venetoclax resistance. In the PB, recent LN emigrants had higher Bcl-XL and Mcl-1 expression than cells immigrating back to the LN. Under ibrutinib treatment, this distinction collapsed, and significantly, the pre-treatment profile reappeared in patients who relapsed on ibrutinib. In response to venetoclax however, Bcl-2 members displayed an early increase, underlining the different modes of action of these two drugs. Profiling by BH3 mimetics was performed in CLL cells fully resistant to venetoclax due to CD40-mediated induction of Bcl-XL, Mcl-1 and Bfl-1. Several dual or triple combinations of BH3 mimetics were highly synergistic in restoring killing of CLL cells. Lastly, we demonstrated that pro-apoptotic Bim interacts with anti-apoptotic Bcl-2 members in a sequential manner: Bcl-2>Bcl-XL>Mcl-1>Bfl-1. Combined, the data indicate that Bcl-XL is more important in venetoclax resistance than Mcl-1 and provide biological rationale for potential synergy between ibrutinib and venetoclax.

In chronic lymphocytic leukemia (CLL), the lymph node (LN) microenvironment delivers critical survival signals by inducing the expression of anti-apoptotic Bcl-2 members Bcl-XL, Bfl-1, and Mcl-1, resulting in apoptosis blockade. We determined previously that resistance against various drugs, among which is the clinically applied BH3 mimetic venetoclax, is dominated by upregulation of the anti-apoptotic regulator Bcl-XL. Direct clinical targeting of Bcl-XL by, e.g., Navitoclax is however not desirable due to induction of thrombocytopenia. Since the actual regulation of Bcl-XL in CLL in the context of the LN microenvironment is not well elucidated, we investigated various candidate LN signals to drive Bcl-XL expression. We found a dominance for NF-κB signaling upon CD40 stimulation, which results in activation of both the canonical and non-canonical NF-κB signaling pathways. We demonstrate that expression of Bcl-XL is first induced by the canonical NF-κB pathway, and subsequently boosted and continued via non-canonical NF-κB signaling through stabilization of NIK. NF-κB subunits p65 and p52 can both bind to the Bcl-XL promoter and activate transcription upon CD40 stimulation. Moreover, canonical NF-κB signaling was correlated with Bfl-1 expression, whereas Mcl-1 in contrast, was not transcriptionally regulated by NF-κB. Finally, we applied a novel compound targeting NIK to selectively inhibit the non-canonical NF-κB pathway and showed that venetoclax-resistant CLL cells were sensitized to venetoclax. In conclusion, protective signals from the CLL microenvironment can be tipped towards apoptosis sensitivity by interfering with non-canonical NF-κB signaling.