Page 201 - Haematologica April 2020

HIF-1α in TP53-disrupted CLL cells
Introduction
Chronic lymphocytic leukemia (CLL) patients with high-risk genomic features such as disruption of the TP53 gene [i.e. del(17p) and TP53 mutations] respond poorly to chemoimmunotherapy and frequently relapse.1-9 Significant advances have been made in the treatment of CLL following the introduction of Bruton tyrosine kinase (BTK) inhibitors.10 Ibrutinib, which is currently approved for the front-line treatment of CLL, induces long-lasting responses in the majority of patients, improving outcome with relatively limited toxicities.10 However, patients with disruption of the TP53 gene (TP53dis) treated with ibrutinib are still characterized by a poorer outcome.11
Hypoxia inducible factor 1 (HIF-1) is an essential regula- tor of cell adaptation to hypoxia and is often up-regulated in tumors due to intratumoral hypoxia or activation of oncogenic pathways.12,13 In tumors, HIF-1 fosters different tumor-promoting mechanisms, including metabolic adap- tation, neoangiogenesis, cell survival and invasion.14
HIF-1 is a heterodimer, which consists of a constitutive- ly expressed HIF-1β subunit and an inducible HIF-1α sub- unit. Besides its traditional regulation via proteasomal degradation, other signaling pathways, such as PI3K/AKT and RAS/ERK1-2, contribute to HIF-1α accumulation, via stability regulation or synthesis induction.12,15
HIF-1α is constitutively expressed in CLL cells com-
pared to normal B cells due to microRNA-mediated down-
regulation of the von Hippel-Lindau protein (pVHL),16 a
ubiquitin ligase responsible for HIF-1α degradation.12 In
addition, in CLL cells, HIF-1α is up-regulated by interac-
tions with stromal cells (SC) and by exposure to hypoxic
microenvironments, thus promoting the survival and
propagation of leukemic cells, and their metabolic adapta-
tion to the protective conditions of the tumor niche.17-20
We have already reported that HIF-1α is involved in drug
resistance mechanisms in patients with unmutated (UM)
immunoglobulin heavy chain variable region genes
(IGHV).20 The TP53 gene encodes one of the best-studied
tumor suppressor proteins, which is often mutated in can-
cer, thus promoting cell survival, proliferation and drug
resistance.21 p53 may also play a pivotal role in the regula-
tion of HIF-1α, since in conditions of prolonged
hypoxia/anoxia, the protein accumulates and promotes
HIF-1α destruction.22 In solid tumors, loss of TP53 func-
tion associates with constitutive elevated levels of HIF-1α.12,22,23
In this study, we found that HIF-1α is over-expressed in CLL cells from patients carrying TP53 aberrations, also elucidating the molecular mechanisms implicated in the constitutive (TP53-related) and inducible (hypoxia- and SC-induced) HIF-1α upregulation. In addition, we observed that the HIF-1α inhibitor BAY87-2243 exerts potent anti-tumor functions, overcoming the constitutive fludarabine resistance of TP53-disrupted CLL cells, and eliciting a strong synergistic cytotoxic effect in combina- tion with ibrutinib.
Methods
Patients’ samples
A total of 102 patients with CLL, diagnosed according to the International Workshop on CLL-National Cancer Institute guidelines,24 were included in the study [40 TP53dis and 62 TP53-
wild type (TP53wt) cases] (Online Supplementary Table S1). Healthy donors’ (HD, n=2) samples were provided by the local blood bank. Patients were untreated or off-therapy for at least 12 months before sampling of peripheral blood (PB) for the experi- ments. Samples were collected after patients’ informed consent in accordance with the Declaration of Helsinki and after approval by the local Institutional Review Board. PB mononu- clear cells (PBMC) were isolated and characterized as detailed in the Online Supplementary Appendix.
Cell lines
The Burkitt’s lymphoma cell line, Séraphine, and the mantle cell lymphoma cell line, Granta-519, were kindly provided by T. Zenz. The TP53wt and the CRISPR/Cas9-mediated TP53 knock- out version (TP53ko) of Granta-519 and Séraphine cell lines were used in the study. The M2-10B4 murine SC line (ATCC #CRL- 1972) was also used. Cell lines were maintained as reported in the Online Supplementary Appendix.
Animals
C57BL/6 Eμ-TCL1 mice were maintained in specific pathogen- free animal facilities and treated in accordance with European Union and Institutional Animal Care and Use Committee (number 716) guidelines. Splenic cells (5x106) were injected intraperitoneally into syngeneic C57BL/6 mice, and experiments were performed with groups of 4-6 mice. Leukemic mice were treated when tumor cells reached 10% in PB. BAY87-2243 was administered at 4 mg/kg in ethanol/solutol/water solution once daily by oral gav- age. Mice were sacrificed at the end of treatment.
Cell culture
In selected experiments, CLL cells were cultured in the pres- ence or absence of M2-10B4 SC, and exposed to PD98059 (Sigma Aldrich, Milan), Y27632 (Sigma Aldrich) or LY249002 (Sellekchem, Houston, TX, USA) for 48 hours (h). CLL cells were exposed for 48 h to BAY87-2243 (Sellekchem); 2-Fluoroadenine- 9-β-D-arabinofuranoside (F-ara-A, Sigma Aldrich); ibrutinib (Sellekchem) used alone or in combination, at the indicated con- centrations. Culture conditions were normoxia or mild hypoxia (1% O2), 5% CO2 at 37°C.
Western blot
Full details can be found in the Online Supplementary Appendix together with the list of antibodies used for western blot (WB) analyses.
Quantitative real-time polymerase chain reaction
Full details of quantitative real-time polymerase chain reac- tion (qRT-PCR) experiments can be found in the Online Supplementary Appendix together with the list of primer sequences.
Gene set enrichment analysis
Gene set enrichment analysis (GSEA, http://www.broad.mit. edu/gsea/index.jsp) was performed as previously described.25,26 Gene sets were assessed as significantly enriched in one of the phenotypes if the nominal P-value and the false discovery rate (FDR)-q value were <0.05.
RHOA and RAS, ERK1-2, AKT and RHOA kinase activity
The isoprenylated membrane-associated RAS or RHOA pro- teins and the non-isoprenylated cytosolic forms were detected as previously described.20 Details of measurement of ERK1-2, AKT and RHOA kinase activity are reported in the Online Supplementary Appendix.
haematologica | 2020; 105(4)
1043