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EPR imaging assessment of reduced tumor oxygenation induced by HIF-2a inhibitor Belzutifan in VHL-deficient renal cell carcinoma models
Kazumasa Horie1, Shun Kishimoto1, Kota Yamashita1, Kazutoshi Yamamoto1, Jeffery R Brender1, Nallanthamby Devasahayam1, Hellmut Merkle2, Murali C Krishna1, and W Marston Linehan1
1NCI, Bethesda, MD, United States, 2NINDS, Bethesda, MD, United States

Synopsis

Keywords: Preclinical Image Analysis, Electron Paramagnetic Resonance

Motivation: In the previous study, EPR oximetry unveiled physiological responses to a VEGF inhibitor, but the potential of EPR oximetry in upstream inhibition of the HIF pathway remained unclear.

Goal(s): To determine if Belzutifan, a HIF-2α inhibitor, affects tumor oxygenation as measured by EPR oximetry and to offer a non-invasive biomarker of its therapeutic efficacy.

Approach: Three xenograft models of clear cell carcinoma were treated with Belzutifan and changes in tumor oxygen levels were assessed using EPR oximetry.

Results: We observed significant alterations in tumor oxygenation exclusively in responding tumors, which we attributed to diminished VEGF expression.

Impact: This research underlines the significance of EPR oximetry as a non-invasive tool to detect early responses to HIF-2α inhibitors like Belzutifan, which could profoundly affect the management and therapeutic approach for cancer treatment, guiding personalized medicine.

Introduction

HIF is a transcription factor that helps regulate the body's response to hypoxia1. In von Hippel-Lindau (VHL) disease-associated renal cell carcinoma, mutations in the VHL gene lead to the stabilization and accumulation of HIF, even in normal oxygen conditions2. This abnormal HIF activity can contribute to the growth of blood vessels and promote VHL-nagative RCC tumor. Belzutifan, a novel HIF-2a inhibitor, is primarily used for the treatment of VHL-negative RCC3,4. Belzutifan specifically targets HIF-2α, one of the subunits of the HIF complex, reducing the production of proteins that drive tumor growth, nutrient uptake, and blood vessel formation, ultimately suppressing the progression of VHL-negative RCC. Belzutifan's mechanism of action may result in tumor shrinkage or the prevention of tumor growth, which can improve the clinical outcome for patients with VHL-negative RCC. In the earlier research, EPR oximetry revealed physiological reactions to a VEGF inhibitor5, but the effect of inhibiting the HIF pathway upstream was not investigated. Consequently, in this present study, our objective is to identify the early responses to Belzutifan by combining EPR oximetry with other physiological MRI imaging techniques.

Method

In this study, we established xenograft models of clear cell renal cell carcinoma (RCC) in nude mice by implanting 5x106 tumor cells (786-O, UOK220, and UOK121 cell lines) subcutaneously into the hindleg. The mice were divided into two groups: the Belzutifan treatment group and the control group. The treatment group received a daily dose of 3 mg/kg of Belzutifan, while the control group was administered a 1% DMSO/PBS solution over a 14-day period.To assess tissue oxygen levels (pO2), we utilized Electron Paramagnetic Resonance Imaging (EPRI). This involved administering the exogenous trityl radical probe, Ox071, via tail vein injection (1.125mmol/kg bolus). Signals were collected through free induction decay (FID) following radiofrequency excitation pulses, with data encoded in three dimensions (x, y, and z gradients) to create 3D spatial maps. T2* maps were generated by analyzing the FIDs, providing EPR line width maps, which were then used to estimate tissue pO2 on a pixel-wise basis. Imaging parameters included an 8.0 μs repetition time and 4000 averages.Dynamic Contrast-Enhanced MRI (DCE-MRI) was carried out using a 1 T scanner, capturing T1-weighted fast low-angle shot (FLASH) images. Imaging parameters consisted of TR = 117.2 milliseconds, TE = 6 milliseconds, flip angle = 30˚, 0.22 × 0.22 mm resolution, 15-second acquisition time per image, and 45 repetitions. Gadolinium-DTPA (4 mL/g of body weight, 50 mmol/L concentration) was injected via a tail vein cannula one minute into the dynamic FLASH sequence. Local Gd-DTPA concentrations were determined using T1 maps derived from six sets of Rapid Imaging with Refocused Echoes (RARE) images acquired with different TR values (320, 400, 600, 1,000, 2,000 and 3,000 ms) before the FLASH sequence.Both EPRI and DCE-MRI scans were performed at the start of the study (Day 0) and repeated on the fourth day of treatment (Day 4).

Results and Discussion

All three xenograft models exhibited varying responses to Belzutifan treatment. Among them, 786-O tumors displayed the most pronounced treatment effect, while UOK220 tumors showed a comparatively weaker response, and UOK121 tumors did not respond at all (Figure 1). Using these models, we assessed the reduction in pO2 after four days of treatment in the Belzutifan treatment group compared to the vehicle treatment group. (Figure 2) As expected, a significant difference was observed in 786-O tumors (Belzutifan group: -3.27 mmHg, vehicle group: -0.13 mmHg; p = 0.05), indicating a substantial impact. In contrast, no significant difference was detected in the UOK121 group (Belzutifan group: -2.11 mmHg, vehicle group: 0.41 mmHg; p = 0.17). However, the anti-angiogenic impact was not verified through perfusion and permeability evaluations conducted with DCE MRI. (Figure 3) The discrepancy was considered to be the result of limited sensitivity of DCE MRI in detecting the weak anti-angiogenic effect, and there may be other factors contributing to the suppression of tumor growth. We assessed the impact of Belzutifan on glucose uptake by analyzing Glut-1 expression through Western blotting. The results indicated reduced Glut-1 levels in both Belzutifan-sensitive 786-O tumors and Belzutifan-resistant UOK121 tumors. (Figure 4) This indicates that the observed tumor growth suppression effect cannot be solely attributed to reduced glucose uptake. Apart from its role in angiogenesis and nutrient uptake, the HIF-2 pathway is also associated with promoting tumor proliferation by upregulating cyclin D1 and genes related to stemness and anti-apoptotic properties. We plan to investigate the contribution of these mechanisms in our ongoing research.

Acknowledgements

This research was supported by intramural funds from the Center for Cancer Research of the National Institutes of Health. The authors declare no conflicts of interest.

References

  1. Ivanova IG, Park CV, Kenneth NS. Translating the Hypoxic Response-the Role of HIF Protein Translation in the Cellular Response to Low Oxygen. Cells. 2019 Feb 1;8(2):114.
  2. Hoefflin, R., Harlander, S., Schäfer, S. et al. HIF-1α and HIF-2α differently regulate tumour development and inflammation of clear cell renal cell carcinoma in mice. Nat Commun 11, 4111 (2020)
  3. Jonasch E, Donskov F, Iliopoulos O, Rathmell WK, Narayan VK, Maughan BL, Oudard S, Else T, Maranchie JK, Welsh SJ, Thamake S, Park EK, Perini RF, Linehan WM, Srinivasan R; MK-6482-004 Investigators. Belzutifan for Renal Cell Carcinoma in von Hippel-Lindau Disease. N Engl J Med. 2021 Nov 25;385(22):2036-2046.
  4. Toledo RA, Jimenez C, Armaiz-Pena G, Arenillas C, Capdevila J, Dahia PLM. Hypoxia-Inducible Factor 2 Alpha (HIF2α) Inhibitors: Targeting Genetically Driven Tumor Hypoxia. Endocr Rev. 2023 Mar 4;44(2):312-322.
  5. Matsumoto S, Saito K, Takakusagi Y, Matsuo M, Munasinghe JP, Morris HD, Lizak MJ, Merkle H, Yasukawa K, Devasahayam N, Suburamanian S, Mitchell JB, Krishna MC. In vivo imaging of tumor physiological, metabolic, and redox changes in response to the anti-angiogenic agent sunitinib: longitudinal assessment to identify transient vascular renormalization. Antioxid Redox Signal. 2014 Sep 10;21(8):1145-55.

Figures

Tumor growth patterns in 786-O, UOK121, and UOK220 after daily Belzutifan treatment (3 mg/kg, blue circles) compared to a control (1% DMSO/PBS, red squares). Tumor sizes were measured every other day by MRI, with the mean tumor volumes and SEM shown. The number of samples is noted in the legend, and treatment cessation is marked by a dashed line on day 14.

EPR Imaging of Tumor Oxygenation Pre- and Post-Belzutifan Treatment. Top Panels: pre- and post-treatment (Day 4) EPR imaging of intra-tumor oxygen levels (pO2) in tumors treated with Belzutifan (3 mg/kg/day), across three models: 786-0, UOK121, and UOK220. The Bottom Bar Plots: the average change in tumor pO2 for each model. Individual tumor data points were included. Decreases in pO2 are indicated by negative values. Data are shown as mean ± SEM.

DCE-MRI data for 786-O, UOK121, and UOK220, comparing perfusion (top row, AIF/min) and permeability (bottom row, AIF/10min) between control (red) and Belzutifan-treated (blue) groups. "ns" indicates no significant differences were observed. Data are shown as mean ± SEM

Expression of GLUT1 in response to Belzutifan treatment. Western blot analysis (left) shows GLUT1 protein levels in 786-O, UOK121, and UOK220 cell lines with (+) or without (-) 5μM Belzutifan treatment. HPRT was used as a loading control. The densitometry analysis of GLUT1 expression normalized to HPRT is presented (right). Data are represented as mean ± SEM. The numerical values atop the bands in the Western blot correspond to the fold change in expression relative to the untreated tumor set as 1.

Proc. Intl. Soc. Mag. Reson. Med. 32 (2024)
4538
DOI: https://doi.org/10.58530/2024/4538