BACKGROUND: Tumor cell survival critically depends on heterotypic communications with non-malignant cells in the microenvironment. Most of these signals converge on the activation of the transcription factor NF-κB that regulates complex cellular functions, including apoptosis, cell survival and proliferation. Even if NF-kB is constitutively active in most malignancies, including chronic lymphocytic leukemia (CLL), and plays a major role in tumorigenesis, there are no currently approved drugs to target it. IT901 has been recently reported as a novel NF-kB inhibitor, showing efficacy in a non-tumor context1.
AIM OF THE WORK: The aim of this work is to test the efficacy of IT901 in CLL and in its more aggressive transformation, Richter syndrome (RS), which represents an unmet therapeutic need. The molecular mechanisms of action of IT901 in leukemic cells are studied, alongside its effects on cells belonging to CLL microenvironment.
RESULTS: IT901 induces apoptosis in primary leukemic cells in a dose- and time-dependent manner, showing significant efficacy after 24h of treatment. The apoptotic response is independent of the prognostic subgroup. Conversely, IT901 has minimal impact upon normal B cells. Treatment of CLL cells with IT901 interferes with NF-kB transcriptional activity, resulting in a diminished binding of both p50 and p65 to DNA. Moreover, biochemical analyses indicate a diminished expression of these subunits in the nucleus, as well as of the whole NF-kB complex in the cytoplasm. At the molecular level, compromised expression of NF-kB triggers activation of the Caspase-3 apoptotic pathway, with increased expression of pro-apoptotic proteins (e.g., Bim), paralleled by a diminished expression of the anti-apoptotic ones (e.g., XIAP). Concomitantly, a prominent increase in mitochondrial ROS is evident, providing a link between IT901 effects and induction of apoptosis.
Recent data reported the involvement of NF-kB as a transcriptional controller of metabolic pathways promoting oxidative phosphorylation in cancer cells. In line with NF-kB constitutive activation in CLL, dynamic measurement of the energetic profile, indicates a reliance on oxidative phosphorylation, with limited glycolytic capacity. After IT901 treatment, there is a dramatic drop in mitochondrial respiration, with compromised ATP production and a net increase in proton leak, suggesting that primary CLL cells are trying to compensate impaired respiration by shifting to glycolysis. This metabolic response is mediated at the transcriptional levels, as IT901 induces a down-modulation of the genes involved in mitochondrial respiration (e.g., ATP5A1) and a concomitant up-modulation of the ones involved in glucose uptake and lactate transport (e.g., GLUT1).
The CLL microenvironment is critical for disease progression and for providing protection from drug-induced apoptosis. Therefore it is important to consider the effects of novel drugs also on non-neoplastic bystander elements. Nurse-like cells (NLC) are a population of monocyte-derived activated macrophages that nurtures CLL cells via soluble and cell contact dependent mechanisms. These interactions are known to activate NF-kB signaling in both partners. Consistently, IT901 inhibited nuclear localization of the p65 subunit in NLC and shifted their polarization towards an M1-phenotype.
These results are confirmed using a xenograft model. The Mec-1 cell line was injected into NSG mice and left to engraft for 2 weeks before beginning treatment. Animals treated with IT901 are characterized by decreased tumor growth and leukemic cells diffusion compared to controls, as shown by a diminished number of leukemic cells in kidneys, liver and spleen.
Finally, IT901 shows promising effects in a small cohort of leukemic cells obtained from RS patients, inducing significant apoptosis by interfering with the expression and nuclear localization of NF-kB.
CONCLUSIONS: Altogether, these results indicate that IT901 blocks NF-kB transcriptional activity. This effect is followed by rapid and marked decrease in genes supporting oxidative phosphorylation, causing mitochondrial damage, ROS release and induction of intrinsic apoptosis. Moreover, IT901 interrupts the support that CLL obtains from the microenvironment. Thus, targeting NF-kB by means of IT901 may be effective for CLL, and possibly even RS patients.
1. Y. Shono et al., Cancer Res76, 377 (Jan 15, 2016).
Furman:Genentech: Consultancy; Janssen: Consultancy; Abbvie: Consultancy, Honoraria; Gilead Sciences: Consultancy; Pharmacyclics: Consultancy, Speakers Bureau.
Asterisk with author names denotes non-ASH members.