HIF-1α Upregulation in TP53 Disrupted Chronic Lymphocytic Leukemia Cells and Its Potential Role As a Therapeutic Target

Background: In chronic lymphocytic leukemia (CLL), disease aggressiveness and drug responsiveness can be ascribed to intrinsic genetic features of the tumor cells, such as TP53 disruption, and to interactions of CLL cells with stromal cells (SC) of the tumor microenvironment. The transcription factor HIF-1α is critically involved in the regulation of genes implicated in key cellular processes, such as cell survival and tumor progression, and also modulates the interactions of CLL cells with SC. HIF-1α expression and transcriptional activity depend on genetic alterations of tumor suppressor genes (e.g. TP53), and on extrinsic signals such as oxygen deprivation and soluble factors. In CLL cells, HIF-1α is active even in normoxia, and its expression is rapidly elevated during hypoxia. We have already reported that HIF-1α activity in CLL cells is upregulated by SC, via activation of Akt, and Ras/ERK1-2 and RhoA/RhoA kinase signaling pathways. SC are well known pro-survival factors, which protect CLL cells from spontaneous apoptosis and fludarabine-induced cytotoxicity. The cytotoxic effects of HIF-1α inhibition, and the ability of HIF-1α targeting agents to reverse constitutive or SC-induced fludarabine resistance, need to be investigated.

Aim: The aim of this study was twofold: 1) to unravel the HIF-1α regulatory pathways in CLL cells, also discriminating between TP53 wild type (TP53^wt)and disrupted (TP53^dis), and 2) to evaluate the ability of HIF-1α inhibition to exert a direct cytotoxic effect and potentiate fludarabine cytotoxicity.

Methods: CLL cells were considered TP53^dis when TP53 mutation or 17p deletion (>10%) were present (n=33). Otherwise they were considered TP53^wt(n=49). HIF-1α gene and protein expression were evaluated by RT-PCR and Western Blot. HIF-1α transcriptional activity was evaluated by ELISA in nuclear extracts. CLL cells were cultured alone or with the M2-10B4 SC line under normoxic or hypoxic conditions. CLL cell cultures were exposed to fludarabine (F-ara-A, 10 μM), BAY 87-2243 (BAY, 1 μM), simvastatin (1 μM) or idelalisib (10 μM), used as single agents or in combination. Cell viability was analyzed by AnnexinV/propidium Iodide immunostaining and flow cytometry. Samples were considered resistant to fludarabine when the relative viability of CLL cells exposed to F-ara-A compared to untreated controls was >0.43 (median value for the whole cohort).

Results: TP53^dis CLL cells expressed a constitutively higher amount of HIF-1α protein compared to TP53^wt CLL cells. This upregulation was not due to a higher HIF-1α gene expression, and was associated to a significantly higher transcriptional activity of HIF-1α (p=0.009). We also evaluated the effect of extrinsic factors on the regulation of HIF-1α. We observed a further increase in HIF-1α expression when both TP53^dis and TP53^wt CLL cells were cultured under hypoxia. Similarly, the co-culture of CLL cells with SC further upregulated HIF-1α, via activation of Akt, Ras/ERK1-2 and RhoA/RhoA kinase signaling cascades, in both subsets. These inducing effects were particularly evident in TP53^dis CLL cells. The specific inhibition of HIF-1α with BAY induced a significant reduction in viability of TP53^dis and TP53^wt CLL cells (p<0.001), confirming the role of HIF-1α in maintaining leukemic cells survival. We also evaluated the ability of BAY to sensitize constitutively resistant TP53^dis CLL cells to F-ara-A. Results from these experiments showed a significant impairment of cell viability after dual treatment with BAY and F-ara-A compared to single agents and untreated control (mean viability: 21.8% BAY + F-ara-A, 34.1% BAY, 47.6% F-ara-A and 59.6% controls; p<0.01). In the last set of experiments, we investigated the role of HIF-1α in the microenvironment-mediated resistance to fludarabine. We found that the direct HIF-1α inhibition with BAY, and the targeting of Ras/ERK1-2 and PI3K/Akt signaling pathways using simvastatin or idelalisib were all able to counteract the protection exerted by SC toward F-ara-A-induced cell death, in both TP53^dis and TP53^wtCLL cells (p<0.03).

Conclusions:Our data demonstrate that HIF-1α is constitutively overexpressed by TP53^dis CLL cells compared to TP53^wt, and is positively regulated by hypoxia and SC in both subsets. HIF-1α targeted inhibition results in high cytotoxicity of CLL cells, and is able to circumvent constitutive p53-related and SC-induced resistance to fludarabine.