Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models
Fei Zhou, Lizhi Pang, Yang Liu, Fatima Khan, Peiwen Chen
Fei Zhou, Lizhi Pang, Yang Liu, Fatima Khan, Peiwen Chen
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Research Article Immunology Oncology

Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models

Abstract

Glioblastoma (GBM) is a highly aggressive brain tumor characterized by extensive crosstalk between glioblastoma stem cells (GSCs) and immunosuppressive microglia, with our previous work identifying CLOCK and TFPI2 as key regulators of this interaction. Here, we uncover a ‘symbiotic exclusivity’ pattern between CLOCK and TFPI2, showing that, despite mutually exclusive amplifications, they sustain symbiotic regulatory interactions in GBM. The CLOCK-BMAL1 complex transcriptionally upregulates TFPI2, while TFPI2-driven hypoxia inducible factor 1 α (HIF-1α) signaling activates nuclear factor k B (NF-kB) P65 to upregulate the CLOCK-BMAL1 complex, creating a positive feedback loop to promote stemness, immunosuppression, and tumor progression. Disrupting the CLOCK-TFPI2 interplay through dual inhibition of their downstream effectors reduces GSC stemness and immunosuppressive microglia, activates antitumor immunity, and synergizes with anti-PD1 therapy to achieve complete tumor regression in 50%–62.5% of tumor-bearing mice. This study uncovers a promising therapeutic strategy for a broader subset of patients with GBM with high expression of either CLOCK or TFPI2, and provides a framework for identifying ‘symbiotic exclusivity’ genes in cancer.

Authors

Fei Zhou, Lizhi Pang, Yang Liu, Fatima Khan, Peiwen Chen

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Figure 7

Inhibition of the downstream pathways of the TFPI2-CLOCK signaling loop reduces GSC stemness and immunosuppressive microglia, activates antitumor immunity, and synergizes with anti-PD1 therapy.

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Inhibition of the downstream pathways of the TFPI2-CLOCK signaling loop ...
(A) Schematic diagram depicting the positive feedback loop between TFPI2 and the CLOCK-BMAL1 complex in GSCs and the strategy of dual targeting their downstream signaling pathways to block GSC self renewal. (B) Survival curves of nude mice implanted with 2 × 105 GSC272 and treated with LDHA inhibitor Stiripentol (150 mg/kg, i.p., every other day), JNK inhibitor JNK-IN-8 (30 mg/kg, i.p., daily), STAT3 inhibitor WP1066 (30 mg/kg, i.p., daily), or Stiripentol in combination with JNK-IN-8 or WP1066 for 2 weeks beginning at day 7. n = 8 mice per group. (C–F) Representative images and quantification of IHC staining for SOX2 (C and D) and CD133 (E and F) in GSC272 tumors from mice with indicated treatments. Scale bar: 100 μm. n = 4. (G and H) Representative images (G) and quantification (H) of flow cytometry for the percentage of CD45lowCD11b+TMEM119+CD206+ microglia (out of CD45lowCD11b+TMEM119+ microglia) in tumors from 005 GSC-bearing mice with indicated treatments. n = 3. (I and J) Representative images (I) and quantification (J) of flow cytometry for the percentage of CD45+CD3+CD8+IFNγ+ T cells (out of CD45+CD3+CD8+ T cells) in tumors from 005 GSC-bearing mice with indicated treatments. n = 3. (K) Survival curves of C57BL/6J mice implanted with 2 × 105 005 GSCs and treated with Stiripentol, JNK-IN-8, or WP1066, and Stiripentol in combination with JNK-IN-8 or WP1066 for 2 weeks beginning at day 7, and with anti-PD1 (10 mg/kg, i.p.) treatment on days 11, 14, and 17. n = 8 mice per group. Data from multiple replicates are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, 1-way ANOVA test (D, F, H, and J), log-rank test (B and K).