Page 202 - Haematologica April 2020

V. Griggio et al.
Cell viability assay
Cell viability was evaluated by flow cytometry using Annexin- V/Propidium Iodide (Ann-V/PI) staining with the MEBCYTO- Apoptosis Kit (MBL Medical and Biological Laboratories, Naka-ku Nagoya).
Statistical analysis
GraphPad Prism (version 6.01, San Diego, CA, USA) was used to perform paired and unpaired t-test, and to calculate Pearson cor- relation coefficient. Results are expressed as mean±standard error of mean (SEM), unless otherwise specified. Statistical significance was defined as a P<0.05. Combination analysis was performed using Compusyn software; combinations were considered syner- gistic when the combination index (CI) was <1.
Results
HIF-1α is over-expressed in chronic lymphocytic leukemia cells from TP53dis patients and in TP53 knockout lymphoma cell lines
Expression levels of HIF-1α protein were comparatively evaluated in HD CD19+ cells, and in CLL cells isolated from TP53dis and TP53wt samples. As expected, HD CD19+ B cells did not express HIF-1α at the baseline normoxic conditions (data not shown). In contrast, leukemic cells from CLL patients exhibited detectable cytosolic and nuclear HIF-1α protein (Figure 1A). Interestingly, CLL cells from patients carrying TP53 abnormalities (TP53dis CLL cells) had significantly higher amounts of the cytosolic and nuclear fractions of HIF-1α subunit, as well as higher HIF-1A mRNA levels compared to CLL cells isolated from TP53wt cases (TP53wt CLL cells) (Figure 1A and B). We eval- uated an enlarged cohort of cases and observed that the association between the expression of HIF-1α and the TP53 status was not influenced by the IGHV mutational status (Online Supplementary Figure S1). The transcriptional activity of HIF-1α was evaluated through the expression of selected target genes.13,15,27 We found a higher expression of GLUT1 and ENO1 in TP53dis CLL cells, compared to TP53wt samples (Figure 1C and D). To corroborate the finding of an association between HIF-1α expression and TP53 status we exploited cell line models. Interestingly, the expression of HIF-1α protein and mRNA was higher in TP53ko Granta-519 and Séraphine lymphoma cell lines, compared to the p53wt (Figure 1E and F). In line with this finding, expression of VEGF, GLUT1 and ENO1 was also significantly higher in TP53ko than in TP53wt Granta-519 and Séraphine cell lines (Figure 1G).
To further investigate the link between TP53 and HIF-1α, we performed GSEA on previously published microarray data from tumor cells isolated from seven TP53dis and 13 TP53wt cases (geocode GSE18971).28 Data of GSEA cases revealed that the TP53 abnormalities were associated with an upregulation of a number of genes belonging to the “GROSS_HYPOXIA_VIA_ELK3_AND_HIF1A_UP” gene set (Figure 2A). The protein ELK3 participates in the tran- scriptional response to hypoxia and controls the expres- sion of several regulators of HIF-1α stability.29 Consistently, the baseline expression of ELK3 was higher in TP53dis compared to TP53wt CLL cells (Figure 2B).
Given its role in HIF-1α regulation,12,30 we also compared pVHL expression in TP53dis and TP53wt samples. Notably, CLL cells from TP53dis patients had reduced amounts of
pVHL compared to TP53wt patients, most likely being responsible for better stabilization of the HIF-1α protein and a repression of its proteasomal degradation in TP53dis cells (Figure 2C). As for HIF-1α expression, there were no differences between pVHL levels according to IGHV mutational status (Online Supplementary Figure S2). These data suggest that TP53 abnormalities lead to a reduced expression of pVHL and subsequently to an accumulation of HIF-1α protein.
Hypoxia and stromal cells further increase HIF-1α expression in chronic lymphocytic leukemia cells from TP53dis and TP53wt patients
We next investigated whether microenvironmental sig- nals, such as oxygen deprivation12 and the interactions with SC,20 had differential effects on HIF-1α according to the TP53 status of the leukemic cells, also in an attempt to better define the underlying molecular mechanisms. To this end, CLL cells were cultured for 48 h in condition of hypoxia or in the presence of SC. Of note, ex vivo culture partially abrogated the TP53-related differential expres- sion of HIF-1α observed at the baseline in freshly isolated CLL cells. In hypoxia, we observed a marked upregulation of the cytosolic and nuclear fractions of HIF-1α protein, which was independent of TP53 status (Figure 3A), and was associated to a reduced expression of pVHL (Figure 3B), and to an activation of the PI3K/AKT and RAS/ERK1- 2 pathways (Figure 3C-F). Consistently, we observed that blocking concentration of pharmacologic agents inhibiting ERK1-2 (PD98059) and PI3K (LY294002) effectively coun- teracted the hypoxia-induced HIF-1α upregulation, inde- pendently of TP53 status (Figure 3G).
In line with previous data,20 we observed a marked upregulation of the cytosolic and nuclear amounts of the HIF-1α when CLL cells were co-cultured with SC (Figure 4A). SC-induced HIF-1α elevation was not associated to a reduced pVHL expression in leukemic cells (Figure 4B), whereas we observed an increased activation of RHOA/RHOA kinase (Figure 4C and D), PI3K/AKT (Figure 4E), and RAS/ERK1-2 (Figure 4F) signaling path- ways. As confirmation, we found that targeted inhibition of ERK1-2, PI3K and RHOA kinase by blocking concentra- tions of pharmacologic agents (i.e. PD98059, LY294002 and Y27632, respectively) effectively counteracted SC- induced HIF-1α upregulation (Figure 4G).
The role of these pathways in modulating HIF-1α over- expression was corroborated by titration experiments showing that exposure of TP53dis and TP53wt CLL cells to increasing concentrations of PD98059, LY294002 and Y27632 induced a progressive reduction of the activity of the targeted kinases, which was associated to a dose- dependent decrease in HIF-1α levels (Online Supplementary Figure S3).
The selective HIF-1α inhibitor BAY87-2243 has anti-tumor activities in chronic lymphocytic leukemia
In line with the role of HIF-1α as a promoting factor for cell survival,12 we found a positive correlation between the baseline levels of HIF-1A mRNA and the 48-h viability of CLL cells during in vitro culture (Figure 5A). Consistently, the viability of leukemic cells isolated from samples char- acterized by baseline HIF-1A mRNA levels above the median value of the entire cohort (HIF-1Ahigh) was signifi- cantly higher than the viability of CLL cells displaying lower HIF-1A values (HIF-1Alow) (Figure 5B and Online
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