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HIF-1α in TP53-disrupted CLL cells
synergistic (CI=0.17) on TP53dis CLL cells and was also evi- dent, although less remarkable, on TP53wt CLL cells (Figure 6C and Online Supplementary Figure S8). Interestingly, we observed that combinations consisting of lower concen- trations of BAY87-2243 + fludarabine were capable of inducing significant reductions in cell viability compared to each drug used as a single agent, and this effect was par- ticularly evident in the TP53dis CLL subset (Figure 6D). The cytotoxic activity of the combinations was also strongly synergistic, as shown by data on CI (Online Supplementary Figure S9). Notably, the significantly higher cytotoxicity of the combination BAY87-2243 + fludarabine was main- tained even when both TP53dis and TP53wt CLL cells were cultured under hypoxia (Online Supplementary Figure S10A and B) or in the presence of SC (Online Supplementary Figure S10C and D).
These results indicate that BAY87-2243 and fludarabine synergistically eliminate primary CLL cells, and that their effect is maintained even in the presence of HIF-1α induc- ing factors that recapitulate the BM niche microenviron- ment.
The combination of BAY87-2243 and ibrutinib exerts a synergistic cytotoxic effect on chronic lymphocytic leukemia cells
Previously reported data indicate that TP53-mutated CLL cells have a lower sensitivity to ibrutinib cytotoxicity in vitro34 and that ibrutinib-induced apoptosis is significant- ly reduced in conditions of hypoxia.19 Therefore, we hypothesized that the combination of an HIF-1α inhibitor and ibrutinib may represent a potentially attractive next step for patients carrying TP53 abnormalities, who are characterized by constitutively higher levels of HIF-1α. Our results show that the combination of BAY87-2243 + ibrutinib determined a significant impairment in the via- bility of TP53dis and TP53wt CLL cells compared to each compound used as a single agent, and was very strongly synergistic (Figure 7A and Online Supplementary Figure S11). Interestingly, similar effects were observed also with lower concentrations of both agents (Figure 7B and Online Supplementary Figure S12). Notably, the combination BAY87-2243 + ibrutinib exerted a significantly higher cytotoxic effect compared to each single compound even when CLL cells from both TP53dis and TP53wt samples were cultured in the presence of SC (Figure 7C).
Overall, these data demonstrate that BAY87-2243 exerts a compelling synergistic effect with ibrutinib, thus provid- ing the rationale for future clinical translation.
Discussion
In this study, we investigated the expression and regula- tion of HIF-1α in TP53dis CLL cells and its potential role as a therapeutic target. We found that CLL cells carrying TP53 abnormalities express significantly higher baseline levels of HIF-1α and have increased HIF-1α transcriptional activity compared to TP53wt cells. Regardless of TP53 sta- tus, the resting levels of HIF-1α are susceptible to further upregulation by microenvironmental stimuli, such as hypoxia and SC. Our data show that HIF-1α is a suitable therapeutic target, the inhibition of which induces a strong cytotoxic effect, capable also of reversing the in vitro fludarabine resistance of TP53dis CLL cells and of exerting synergistic effects with ibrutinib.
Hypoxia has a detrimental role in the pathobiology of several solid and hematologic tumors.35,36 The identifica- tion of new potential targets in CLL is certainly important for high-risk patients, for whom there is still no effective cure, and, in addition, the development of new therapies might be effective in a broader setting of B-cell lympho- proliferative disorders.
It has been previously reported that HIF-1α levels vary considerably among CLL patients and that its overexpres- sion is a predictor of a poor survival.17,37 In line with obser- vations made in solid tumors, where p53 promotes the ubiquitination and proteasomal degradation of the HIF-1α subunit in hypoxia,12,22 we postulated that abnormalities of the TP53 gene might have an influence on the regulation of HIF-1α in CLL cells. To the best of our knowledge, this is the first study examining the differential expression and transcriptional activity of HIF-1α in patients with TP53-deficient CLL, also uncovering new mechanisms for HIF-1α modulation in leukemic cells. Our data show that the high-resting levels of HIF-1α detected in TP53dis sam- ples associate both to an increased transcription, as shown by the higher HIF-1A mRNA levels, and to a decreased degradation, as shown by the higher baseline expression of ELK3 and by the lower pVHL amounts.
We next investigated the cellular pathways implicated in HIF-1α regulation mediated by extrinsic factors. Hypoxia-induced HIF-1α upregulation is not only due to a reduced pVHL-mediated protein degradation, which is a well-known mechanism in condition of oxygen depriva- tion, but also to an increased activation of RAS/ERK1-2 and PI3K/AKT signaling pathways. In contrast, pVHL expression in leukemic cells is not affected by SC, which instead activate the RAS/ERK1-2, RHOA/RHOA kinase and PI3K/AKT intracellular pathways, thus leading to HIF- 1α overexpression. These results endorse recent data that implicate a number of paracrine factors in the transcrip- tional and translational regulation of HIF-1α in different tumor models.38
Previous data have suggested that HIF-1α targeting is a promising therapeutic strategy in CLL, potentially capable of synergizing with chemotherapeutic agents. The only HIF-1α inhibitor that has been preclinically tested in CLL is EZN-2208, a topoisomerase I inhibitor that has also been shown to down-modulate HIF-1α.17 We therefore tested BAY87-2243, a more selective HIF-1α inhibitor that has already shown in vivo anti-tumor efficacy in a lung tumor model, without any signs of toxicity.39 Interestingly, BAY87-2243 demonstrated a direct cytotoxicity towards leukemic cells isolated from CLL patients, and this effect was independent of TP53 status. As far as we know, this is the first evidence of the anti-tumor activity of BAY87- 2243 in hematologic tumors, and particularly in CLL.
Patients with CLL and TP53 abnormalities are intrinsi- cally resistant to fludarabine-based chemotherapy regi- mens.9,31-33,40,41 Our data demonstrate: i) higher baseline lev- els of HIF-1α in fludarabine-resistant CLL cells; and ii) a further upregulation of HIF-1α after exposure to hypoxia and SC. Given this, we investigated the ability of BAY87- 2243 to overcome both intrinsic (i.e. TP53-related) and inducible (i.e. SC-induced) resistance to fludarabine. Our data show that BAY87-2243 potently synergizes with flu- darabine in vitro, and that their combined cytotoxic effect was especially evident in TP53dis samples. Interestingly, HIF-1α inhibition was also effective in overcoming the TP53-independent fludarabine-resistance induced by
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