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Abstract: S868

Type: Oral Presentation

Presentation during EHA23: On Saturday, June 16, 2018 from 16:15 - 16:30

Location: Room K1

Background

Treatment of high-risk chronic lymphocytic leukemia (CLL) patients remains an unmet clinical need. Disease aggressiveness can be ascribed to intrinsic features of the tumor cells (i.e. TP53 disruption) and to the interaction of CLL cells with stromal cells (SC) of the microenvironment. HIF-1 is a transcription factor implicated in cell adaptation to hypoxia and is involved in the regulation of genes implicated in tumor progression. In CLL cells, the α subunit of HIF-1 (HIF-1α) is constitutively expressed even in normoxia and regulates the protective interactions that the leukemic cells establish with the microenvironment.

Aims

The aims of this study were to understand HIF-1α regulatory pathways in CLL cells from TP53 disrupted (TP53^dis) and wild type (TP53^wt) patients, and to evaluate the ability of HIF-1α inhibition to exert synergistic cytotoxic effects in combination with fludarabine and ibrutinib.

Methods

Del(17p) in CLL cells was assessed by fluorescence in situ hybridization and the presence of TP53 gene mutations was evaluated by Sanger sequencing. CLL patients with mutation of the TP53 gene, or >40% del(17p) in the absence of TP53 mutation, were included in the TP53^dis subset. Patients with <10% del(17p) and without TP53 mutation were considered TP53^wt. In selected experiments, CLL cells were cultured in the presence or absence of M2-10B4 SC, and exposed to PD98059, Y27632, LY249002, BAY87-2243, F-ara-A or ibrutinib. Culture conditions were 21% (normoxia) or 1% (hypoxia) O2, 5% CO2 at 37°C. Ras, ERK1-2, Akt, HIF-1α, Elk3 and pVHL expression was evaluated by Western Blot. RhoA and RhoA kinase acitivity was measured by specific immunoassays. HIF-1A, p21 and ENO1 gene expression was assessed by RT-PCR. Cell viability was analyzed by AnnexinV/propidium Iodide immunostaining.

Results

We found that primary CLL cells from patients carrying TP53 abnormalities (TP53^dis CLL cells) had constitutively higher transcriptional activity and expression levels of the α subunit of HIF-1 compared to CLL cells isolated from TP53^wt samples (TP53^wt CLL cells). HIF-1α upregulation detected in the TP53^dis subset was due to a reduced expression of the HIF-1α ubiquitin ligase von Hippel-Lindau protein (pVHL) and more active PI3K/Akt and Ras/ERK1-2 signalling pathways. Hypoxia and SC further enhanced HIF-1α accumulation in both TP53^dis and TP53^wt CLL cells. Hypoxia-mediated HIF-1α upregulation was due to a decreased pVHL expression and to the activation of PI3K/Akt and Ras/ERK1-2 signalling pathways. SC did not affect pVHL expression, but induced an increased activity of Ras/ERK1-2, RhoA/RhoA kinase and PI3K/Akt pathways, leading to HIF-1α accumulation. Interestingly, in vitro fludarabine-resistant CLL cells were mostly TP53^dis and expressed significantly higher levels of HIF-1A mRNA compared to fludarabine-sensitive cells. The HIF-1α inhibitor BAY87-2243 reversed the constitutive fludarabine resistance of leukemic cells isolated from patients carrying TP53 abnormalities, and counteracted the fludarabine resistance induced by SC. BAY87-2243 also elicited a strongly synergistic cytotoxic effect in combination with ibrutinib.

Conclusion

Overall, our data indicate that HIF-1α is overexpressed in CLL cells, especially in the presence of TP53 abnormalities, and is susceptible of positive regulation by hypoxia and SC. From the translational standpoint, HIF-1α can be regarded as a crucial target whose inhibition warrants further evaluation, also in combination with currently available therapies.

Session topic: 5. Chronic lymphocytic leukemia and related disorders – Biology & Translational Research

Keyword(s): Hypoxia-sensing, P53, Targeted therapy