Intrinsic Activation of the NLRP3 Inflammasome in Primary Human ALL Cells Is Regulated By Endogenous HMGB1 and Bone Marrow Macrophages

Inflammasomes are cytosolic signaling complexes that assemble in response to damage-associated molecular patterns (DAMPs). Intrinsic expression of the NLRP3 has been identified in acute lymphoblastic leukemia (ALL) cell lines and implicated in glucocorticoid resistance (Paugh W et al., 2015). The endogenous function of NLRP3 in ALL remains unknown. In this study, we use primary human ALL cells to demonstrate that NLRP3 is activated in primary ALL cells in response to chemotherapy stress, is regulated by the endogenously expressed "danger protein" HMGB1 and can be silenced through direct contact with bone marrow macrophages.

To determine whether chemotherapy treatment of primary ALL cells induces NLRP3 expression, we treated CD19-selected cells from n=3 pediatric patients with ALL and lymphocytes from healthy donors with either 2nM doxorubicin or vehicle and compared NLRP3 expression by qPCR and Western blot at 24 hours. NLRP3 mRNA expression was increased by 4.5 + 0.8- fold over baseline in doxorubicin-treated ALL cells compared to no change in vehicle-treated ALL and undetectable levels in healthy lymphocytes. This corresponded to a 2-fold increase in NLRP3 protein expression was measured by Western Blot. Neither doxorubicin-treated nor vehicle-treated ALL expressed other NOD-family inflammasome proteins, including NLRP1 and NLRC4.

In healthy monocytes, NLRP3 activates caspase-1 to induce transcription of inflammatory cytokines. To determine whether NLRP3 up-regulation has a similar function in primary ALL cells, we measured pro- and cleaved-caspase-1 in doxorubicin-treated primary ALL, vehicle-treated ALL and healthy lymphocytes. Primary ALL cells treated with doxorubicin expressed the transcriptionally active p20 subunit of caspase-1 by Western blot; vehicle-treated ALL cells and healthy lymphocytes did not. This corresponded to a 3.7 + 0.61-fold increase in global caspase-1 transcription by mRNA in doxorubicin-treated ALL cells and a significant increase in soluble IL1B production by ELISA (p<0.02). Treatment of primary ALL with staurosporin (an autophagy-inducer) and serum starvation up-regulated NLRP3 but not caspase 1/p20. Taken together, these data established proof-of-principle that primary ALL cells activate the NLRP3 inflammasome in response to clinically relevant cellular stress.

The activating signal for NLRP3 assembly is unknown in leukemia. We have previously observed that DNA-binding DAMP high mobility group-box 1 (HMGB1) is endogenously expressed in ALL cells. To determine whether HMGB1 activates NLRP in leukemia, we knocked down HMGB1 by siRNA in primary ALL cells (n=3 patients) and measured NLRP3 and caspase-1/p20 at baseline and after treatment with doxorubicin vs. vehicle. HMGB1 siRNA ALL cells displayed at least a 2-fold decrease in NLRP3 protein expression 48 hours after knockdown compared to control siRNA cells that could not be rescued with doxorubicin treatment. Co-immunoprecipitation studies indicate that HMGB1 forms a binding complex in primary ALL cells.

To determine whether NLRP3 expression in primary ALL is impacted by cells in the nonmalignant field, we co-cultured primary ALL cells with bone marrow-derived monocyte/macrophage, either directly or separated by 0.6 uM transwell. Unexpectedly, NLRP3 expression was completely abrogated in primary ALL cells when they were placed in direct, but not transwell, co-culture with macrophages. Direct co-culture of ALL with peripheral monocytes, however, did not abrogate NLRP3. These data indicate that direct interactions of ALL with macrophages in the nonmalignant bone marrow microenvironment have a negative regulatory effect on NLRP3 inflammasome activation.

Taken together these data show, for the first time in primary pediatric ALL cells, that NLRP3 inflammasome is activated in response to chemotherapy cell stress that is regulated by endogenous HMGB1 expression and direct interactions with bone marrow macrophages. Studies are underway to define these interactions and their physiologic consequences for ALL cell survival in tne marrow niche.