JURL-MK1

Activated T Cell-Conditioned Medium Strongly Counteracts Imatinib-Induced Apoptosis of JURL-MK1 Cells, and IFNγ Neutralization Strongly Inhibits This Anti-Apoptotic Effect

Tyrosine kinase inhibitors (TKIs) have revolutionized chronic myeloid leukemia (CML) therapy, yet residual disease persists due to factors within the microenvironment. In particular, TKI treatment increases interferon γ (IFNγ) production, which appears to counteract imatinib-induced apoptosis in CML cells. Ujvari et al. aim to elucidate how IFNγ mediates a STAT1-dependent anti-apoptotic effect, including its role in upregulating survival proteins in CML stem/progenitor cells.

To study the role of IFNγ in the effect of activated T cell secretome on imatinib-treated CML cells, they conducted IFNγ neutralization experiments on the JURL-MK1 CML cell line using conditioned medium from T cells isolated from healthy donor PBMCs (Fig. 1). Imatinib induced high apoptosis rates in JURL-MK1 cells with non-activated T cell medium, but this effect was strongly counteracted by medium from CD3/CD28 bead-activated T cells. Importantly, a neutralizing anti-IFNγ antibody largely reversed the anti-apoptotic effect of activated T cell medium, though not completely. However, simultaneous neutralization of IFNγ and GM-CSF nearly abolished the anti-apoptotic effect of activated T cell medium on JURL-MK1 cells (Fig. 1b), indicating that the anti-apoptotic activity of activated T cell secretome is mainly mediated by IFNγ, with a minor contribution from GM-CSF.

Fig. 1. IFNγ neutralization markedly counteracts the anti-apoptotic effect of activated T cell-conditioned medium on imatinib-treated JURL-MK1 cells (Ujvari D, Malyukova A, et al., 2020).

Montelukast induces Apoptosis in K562 and JURL-MK1 Cells

Chronic myeloid leukemia (CML) is characterized by the Bcr-Abl oncoprotein, and TKIs have limitations in eradicating leukemic stem cells (LSCs) due to aberrant pathways like leukotriene signaling. CysLT1R, a receptor in this pathway, is overexpressed in cancers and targeted by montelukast. Zovko et al. previously showed that montelukast reduces CML cell growth. Here, they aim to further investigate the role of CysLT1R in CML cell survival and the mechanisms by which montelukast induces apoptosis in CML cells.

Treatment with montelukast and imatinib, caused cytosol localization of cytochrome c (Fig. 2A). Release of cytochrome c leads to activation of caspases and cleavage of the caspase-3 substrate PARP-1. Montelukast treatment resulted in PARP-1 protein cleavage in the K562 cells. Ratio of cleaved vs. full length PARP-1 increased in a dose dependent manner (Fig. 2B). Furthermore, they observed a dose-dependent increase in proapoptotic Bax protein signal in K562 cells upon montelukast treatment (Fig. 2B). The growth of JURL-MK1 cells was significantly inhibited by montelukast, and when combined with imatinib the receptor antagonist induced further growth inhibition (Fig. 2C). PARP-1 protein was also cleaved in JURL-MK-1 cells treated with montelukast (Fig. 2D). Montelukast (4 µM) also induced significant caspase-3 activation (Fig. 3A and B). Imatinib, which was used as a positive control for apoptosis induction, activated caspase-3 but when used in combination with 4 µM montelukast, caspase-3 was further activated. Moreover, the caspase inhibitor Z-VAD was able to partially inhibit montelukast-induced cell death (Fig. 3C).

Fig. 2. Montelukast induces apoptosis in K562 and JURL-MK1 cells (Zovko A, Yektaei-Karin E, et al., 2018).

Fig. 3. Caspase-3 activation in the K562 cell line (Zovko A, Yektaei-Karin E, et al., 2018).