Even if NOTCH1 is commonly mutated in chronic lymphocytic leukemia (CLL), its functional impact in the disease remains unclear. Using CRISPR/Cas9-generated Mec-1 cell line models, we show that NOTCH1 regulates growth and homing of CLL cells by dictating expression levels of the tumor suppressor gene DUSP22. Specifically, NOTCH1 affects the methylation of DUSP22 promoter by modulating a nuclear complex, which tunes the activity of DNA methyltransferase 3A (DNMT3A). These effects are enhanced by PEST-domain mutations, which stabilize the molecule and prolong signaling. CLL patients with a NOTCH1-mutated clone showed low levels of DUSP22 and active chemotaxis to CCL19. Lastly, in xenograft models, NOTCH1-mutated cells displayed a unique homing behavior, localizing preferentially to the spleen and brain. These findings connect NOTCH1, DUSP22, and CCL19-driven chemotaxis within a single functional network, suggesting that modulation of the homing process may provide a relevant contribution to the unfavorable prognosis associated with NOTCH1 mutations in CLL.
PD-L1 is expressed by a subset of patients with metastatic melanoma (MM) with an unfavorable outcome. Its expression is increased in cells resistant to BRAF or MEK inhibitors (BRAFi or MEKi). However, the function and regulation of expression of PD-L1 remain incompletely understood.After generating BRAFi- and MEKi-resistant cell lines, we observed marked up-regulation of PD-L1 expression. These cells were characterized by a common gene expression profile with up-regulation of genes involved in cell movement. Consistently, in vitro they showed significantly increased invasive properties. This phenotype was controlled in part by PD-L1, as determined after silencing the molecule. Up-regulation of PD-L1 was due to post-transcriptional events controlled by miR-17-5p, which showed an inverse correlation with PD-L1 mRNA. Direct binding between miR-17-5p and the 3’-UTR of PD-L1 mRNA was demonstrated using luciferase reporter assays.In a cohort of 80 BRAF-mutated MM patients treated with BRAFi or MEKi, constitutive expression of PD-L1 in the absence of immune infiltrate, defined the patient subset with the worst prognosis. Furthermore, PD-L1 expression increased in tissue biopsies after the metastatic lesions became resistant to BRAFi or MEKi. Lastly, plasmatic miR-17-5p levels were higher in patients with PD-L1+ than PD-L1- lesions.In conclusion, our findings indicate that PD-L1 expression induces a more aggressive behavior in melanoma cells. We also show that PD-L1 up-regulation in BRAFi or MEKi-resistant cells is partly due to post-transcriptional mechanisms that involve miR-17-5p, suggesting that miR-17-5p may be used as a marker of PD-L1 expression by metastatic lesions and ultimately a predictor of responses to BRAFi or MEKi.
Key Points• Hypoxia shapes the CLL lymph node microenvironment by acting through the A2A adenosine receptor.• Inhibiting the A2A adenosine receptor counteracts the effects of hypoxia on CLL cells, macrophages, and T lymphocytes. adenosine receptor counteracts these effects on all cell populations, making leukemic cells more susceptible to pharmacological agents while restoring immune competence and T-cell proliferation. Together, these results indicate that adenosine signaling through the A2A receptor mediates part of the effects of hypoxia. They also suggest that therapeutic strategies to inhibit the adenosinergic axis may be useful adjuncts to chemotherapy or tyrosine kinase inhibitors in the treatment of CLL patients.
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