Primed for Death: Hypoxia Sensitizes Primary CLL Cells towards Compounds Targeting Mitochondrial Integrity

CLL cells prefer to remain in the microenvironment since they feel safe. CD40 ligand (CD40L)-CD40 interaction induces proliferative/anti-apoptotic genes in CLL cells, which protects them from apoptosis and most cytotoxic drugs by the microenvironment.

Research interested in identifying novel drugs that effectively target CLL cells within microenvironmental niches has to consider further microenvironmental stimuli, especially hypoxia. Lymph nodes, especially those being infiltrated by malignant cells show low oxygen tension (1%). Prior CLL investigations never took this important factor into account. The impact of hypoxia on survival and drug-resistance is still unknown.

Therefore we have established an in vitro model, which mimics hypoxic conditions and CD40L-CD40 interaction, in order to understand the molecular basis of drug resistance of CLL resident in the microenvironment. CLL cells were cultured on CD40L feeder cells and kept up to 96 hours in hypoxia (1% O₂ tension) or normoxia (21% O₂ tension).

We applied several drugs under these conditions to investigate the differences between normoxia and hypoxia. The miRNA expression was determined by using Illumina Bead Chip Arrays compromising 752 miRNAs. Gene expression was analyzed via mRNA-based Illumina microchip array. Target miRNAs and mRNAs were validated by qRT-PCR. Apoptosis was determined by AnnexinV-7AAD and JC-1 staining (mitochondrial outer membrane permeabilization) and subsequent flow cytometry.

In solid cancers hypoxia is expected to protect malignant cells from chemotherapy. We made similar observations, since classical DNA-targeting drugs were inefficient to kill CLL cells cultured on CD40L feeder cells under hypoxia and normoxia. However, we identified ABT-737, which affect mitochondrial integrity, to be even more efficient under hypoxic conditions and CD40L interaction compared to CD40L stimulation and normoxia (74,1% vs. 52,1% apoptotic cells, n=15; p<0.001). Interestingly, overall survival of primary CLL cells during CD40L-CD40 interaction without any cytotoxic treatment was higher under hypoxia compared to normoxia. To understand this discrepancy, we investigated the expression of several mitochondrial localized anti-/pro-apoptotic genes on RNA and protein level. We identified, that the de-regulation of BCL_XL and MCL1 is crucial for ABT-737 sensitivity during hypoxic conditions. This de-regulation was also detectable during CD40L interaction. BCL_XL deregulation could be attributed to differential NF-κB expression, as determined by EMSA. Since MCL1 protein expression differs from its mRNA expression, we expected regulation prior to protein synthesis. Indeed, we could identify miRNAs, which were upregulated during hypoxia and CD40L stimulation and regulate MCL1 expression. These miRNAs were validated by luciferase expression assays.

Here we investigated for the first time the impact of oxygen tension on therapeutic response of CLL cells. We assume that small molecules like ABT-737, which specifically target mitochondria, might be efficient in targeting CLL cells protected by CD40L-CD40 interaction within the microenvironment.

Development of novel in vitro models will allow us to understand the specific molecular changes induced by microenvironmental stimuli and to develop novel therapeutic targets.

L.P.F. and M.Hue. contributed equally to this work.

No relevant conflicts of interest to declare.