Targeting NLRP3 Inflammasome-Induced Therapy Resistance in ALL

Background: Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood cancer and the most frequent cause of death from cancer before 20 years of age. Survival rates for patients with relapsed ALL remain less than 50% due to the emergence of chemoresistance, particularly steroid resistance. Several lines of evidence suggest the importance of therapy-related activation of the NLRP3 inflammasome in ALL treatment resistance. The NLRP3 inflammasome has been shown to convey steroid resistance, through epigenetic mechanisms leading to NLRP3 promoter hypomethylation and upregulation of NLRP3 expression, and through inflammasome-dependent modulation of the expression of the glucocorticoid receptor.

Objectives: The primary objective of this study is to define the kinetics of activation of the NLRP3 inflammasome in ALL patients following systemic chemotherapy. A secondary objective is to determine whether inhibition of the NLRP3 inflammasome enhances the ALL response to conventional chemotherapy in established, patient-derived xenograft (PDX) preclinical models of pediatric ALL. Specifically, we will investigate whether a newly developed synthetic triterpenoid, CDDO-2P-Im (which we have shown to inhibit activation of NLRP3), can suppress the in vivo growth of steroid-resistant human ALL PDX lines and augment their response to the steroid dexamethasone (DEX), and other conventional chemotherapy agents.

Methods: We obtained 1-3 ml of peripheral blood from newly diagnosed ALL patients (0-40 years of age) before and after induction chemotherapy and from normal controls without significant inflammatory or infectious diseases under the UH Hospital Cleveland Medical Center IRB-approved protocol STUDY20190453. Either the Ella Simple Plex protein assay or ELISA were used to determine the expression levels of inflammasome associated proteins such as IL-1β, IL-18, caspase-1 and ASC. Western blot was used to analyze the inflammasome signaling proteins such as casepase-1, IL-1β, IL-18, ASC, NLRP3, AIM2, and LonP1 in the cell lysates. Cell viability assay with Cell-Titer-Glo was performed with different leukemia cell lines, such CEM-1, CEM-7, NALP-3 to determine effects of CDDO-2P-Im and DEX on the leukemia growth. Flow cytometry with Annex-V combined with 7-AAD staining was used for apoptosis assay to detect the effect of CDDO-2P-Im and chemotherapy reagents such as DEX. Leukemia PDX models were established to evaluate the in vivo effects of DEX combined with CDDO-2P-Im.

Results: Currently 22 ALL patients and 13 controls were included, with plasma samples from 7 ALL patients. The post chemotherapy expression levels of IL-18, which is one of the important inflammasome proteins in the plasma, are significantly elevated compared with pre-chemotherapy. The expression of NLRP3 mRNA and that of downstream proteins caspase-1 and IL-18 in ALL culture supernatants are upregulated after leukemia cells are exposed to doxorubicin and dexamethasone at 12 and 18 hours. The newly synthesized triterpenoid analog CDDO-2P-Im, which inhibits the activation of NLRP3 inflammasome, inhibited the in vitro growth of ALL cells in culture, induced apoptosis of both steroid-sensitive and steroid-resistant leukemia cells, enhanced the anti-leukemia effect of dexamethasone in steroid-resistant leukemia cells and significantly prolonged the survival of mice bearing human PDX lines.

Conclusions: Our preliminary data indicate that chemotherapy activates the NLRP3 inflammasome and downstream signals in patients undergoing therapy for ALL. Importantly, the novel triterpenoid analog, CDDO-2P-Im blocks NLRP3 activation, suppresses ALL viability and exhibits significant single agent activity in steroid-resistant ALL.