The Hypoxia-Inducible Factor-1alpha Is Constitutively Upregulated in TP53 Disrupted CLL Cells: A Potential Target to Overcome Fludarabine Resistance

BACKGROUND: Treatment of patients with fludarabine-resistant chronic lymphocytic leukemia (CLL) is an unmet clinical need. Fludarabine resistance in CLL can be ascribed to intrinsic genetic features of the tumor cells, mainly consisting in TP53 disruption, and to the interactions of CLL cells with stromal cells (SC) of the tumor microenvironment. One of the main players of SC-induced fludarabine resistance is the CXCL12/CXCR4 axis, which activates in CLL cells the Ras/ERK1-2 and RhoA-dependent signaling pathways. To be active transducers Ras and RhoA need to undergo isoprenylation by means of small molecules produced by the mevalonate (Mev) pathway. We reported that the Mev pathway-regulated Ras and RhoA signaling cascades and the downstream hypoxia inducible factor (HIF)-1α/P-glycoprotein axis are more active in IGHV unmutated than in mutated CLL cells, leading to a constitutive protection from doxorubicin-induced cytotoxicity. HIF-1α is a transcription factor constitutively expressed in CLL cells and controls the expression of several genes implicated in survival and cell metabolism. The Mev pathway manipulation with simvastatin (Sim), as well as the targeted inhibition of ERK1-2, RhoA kinase and HIF-1α, restore the sensitivity of CLL cells to doxorubicin. The role of the Mev pathway, the Ras and RhoA signaling, and the transcription factor HIF-1α in regulating the SC-induced fludarabine resistance of TP53 disrupted (TP53^dis) CLL cells is currently unknown.

AIM: The aim of this study was to evaluate the activity of the Mev pathway-regulated Ras and RhoA signaling cascades and the transcription factor HIF-1α in TP53^dis CLL cells, in order to identify biochemical and molecular targets potentially amenable to therapeutic intervention.

METHODS: Purified CLL cells were cultured alone or with the M2-10B4 SC line. Cell cultures were exposed to fludarabine (10 μM), Sim (1 μM) or the HIF-1α inhibitor YC-1 (10 μM). The activity of the Mev pathway was measured by quantification of [³H]acetate-labeled cholesterol and farnesyl pyrophosphate produced by CLL cells. Ras and RhoA activities were evaluated by pull-down assay and ELISA, respectively. ERK1-2 phosphorylation was evaluated by Western Blot. RhoA kinase, Akt and HIF-1α activities were measured with specific immunoassay. The glucose flux through glycolysis and tricarboxylic acid cycle was measured by radiolabeling cells with [6-¹⁴C]-glucose and quantifying the amount of glucose transformed into CO₂. Cell viability was analysed by Annexin-V/propidium Iodide immunostaining and flow cytometry. Patients with TP53 mutation or 17p deletion were considered TP53^dis.

RESULTS: Co-culture with SC upregulated the Mev pathway activity of CLL cells, activated the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling, and increased the transcriptional activity of HIF-1α. Blocking the Mev pathway by Sim significantly reduced the SC-induced upregulation of Ras and RhoA signalling pathways, and counteracted the SC-mediated protection of CLL cells from fludarabine-induced cytotoxicity. Based on these results we evaluated the activity of the Mev pathway, and the Ras and RhoA signalling in TP53^dis and TP53 wild-type (TP53^wt) CLL cells. There was no difference between the two groups in terms of Mev pathway activity, Ras/ERK1-2 and RhoA/RhoA kinase signal transduction, and Akt activity. By contrast, we found that TP53^dis CLL cells had a significantly higher transcriptional activity of HIF-1α than TP53^wt CLL cells (p=0.02). CLL patients carrying a percentage of 17p deletion >60% in the tumor clone had the higher values of HIF-1α activity. As a result, TP53^dis CLL cells overexpressed the glycolytic HIF-1α target gene α-enolase and had a more active glycolytic activity than TP53^wt CLL cells (p=0.003). In TP53^dis CLL cells, Sim significantly inhibited the Mev pathway, and the Ras and RhoA signalling, but had no effect on the upregulated transcriptional activity of HIF-1α and on the protective effect exerted by SC toward fludarabine-induced cytotoxicity. Conversely, the HIF-1α inhibitor YC-1 partially restored the fludarabine sensitivity of TP53^dis CLL cells, as shown by its ability to counteract the protective effect exerted by SC toward fludarabine-induced cell death.

CONCLUSIONS: Our data demonstrate that the targeted inhibition of HIF-1α is a promising strategy to circumvent the SC-induced fludarabine resistance of TP53^dis CLL cells.