The DNA-Binding Protein High Mobility Group Box 1 (HMGB1) Interacts with RelB to Maintain Non-Canonical NF-κb Signaling, Cell Survival and Immune Evasion in Infant Acute Lymphoblastic Leukemia

Disordered NF-κB signaling is a hallmark of many hematologic malignancies but has not been well-studied in MLL-rearranged infant lymphoblastic leukemia (iMLL-ALL), a subtype of pediatric ALL with an extremely poor prognosis in need of new therapy. Given the stable genome and short latency of iMLL-ALL, targeting of oncoproteins that play a coordinated role in regulation of the leukemic transcriptome represent ideal targets. Here, we present one such potential target: a complex formed between the nucleosome-stabilizing protein high mobility group box 1 (HMGB1) and RelB, a transcriptionally active subunit of the noncanonical NF-κB pathway.

Disruptions in the non-canonical NF-κB pathway, which normally functions to support lymphogenesis, are known to drive lymphoid malignancies. Specifically, changes in proteasomal processing of the p100 subunit modulate nuclear p52/RelB regulation of the NF-κB2 gene. In stable short-term culture, we observed that primary infant MLL-ALL cells (derived from the diagnostic pheresis specimen of a 9 month old with t(4;11)) express an endogenous NF-κB subunit profile consistent with constitutive non-canonical activation, including significantly increased levels of IKKa, RelB, and NF-κB2 by Western Blot and qPCR compared to healthy human B cells and cytogenetically normal ALL. First, we asked whether exogenous "drivers" of the canonical (p65/RelA) pathway could alter this expression profile by treating iMLL-ALL with TNFalpha and bacterial LPS. Exogenous cytokine treatment did not alter expression levels of IKKa, RelB or NF-κB2, nor did it increase p65RelA, likely due to the lack of pattern recognition receptors on precursor lymphoblastic cells.

We have previously demonstrated that infant MLL-ALL express high intracellular levels of the nucleosome-stabilizing protein high mobility group box-1 (HMGB1). During infection, HMGB1 forms a transcription regulatory complex with RelB that, by epigenetic means, silences inflammatory cytokine promoters as a negative feedback. In this study, we hypothesized that HMGB1-RelB interactions stabilize and maintain a pro-leukemic NF-κB program. Because the function of HMGB1 is determined by post-translational modifications, we first verified the DNA-binding disulfide form of the protein in the nuclear fraction of iMLL-ALL cells by immunoprecipitation. Next, we proved a physiologically relevant interaction between HMGB1 and RelB by co-immunoprecipitation; both HMGB1 and RelB could be identified by immunoblot from iMLL-ALL extracts probed for either for RelB or HMGB1. Detection of the complex required minimal stabilization, suggesting a durable interaction. Preliminary studies suggest that the complex associates with the acetylated form of histone 3 (H3), rather than histone 1 as in sepsis models.

We then determined the effect of HMGB1 siRNA knockdown on the NF-κB subunit expression profile, proliferation and survival of iMLL-ALL, nonleukemic control PBMC and 293T cells. HMGB1 siRNA produced 30-50% spontaneous apoptosis in MLL-ALL, but not nonleukemic control cells, measured by Annexin/PI. iMLL-ALL transfected with a nonsense siRNA sequence did not apoptose. HMGB1 siRNA also silenced expression of RelB and NF-κB 2, measured by Western Blot and qPCR. Unexpectedly, HMGB1 siRNA restored expression of critical canonical NF-κB subunits: p65/RelA, the TNF-associated factor TRAF3, and the proteasome regulator NEMO. Further, HMGB1 siRNA knockdown resulted in a 51.3% (+/- 3.47, p<0.0001) increase in engulfment by activated bone marrow macrophages in a dual-label phagocytosis assay, compared to nonsense siRNA and control iMLL-ALL- suggesting that HMGB1 expression plays a role in evasion of iMLL-ALL by the innate immune system.

Taken together, these data indicate that HMGB1-RelB interactions represent an important regulatory component of the NF-κB signaling program in human infant MLL-ALL cells that support survival and leukemogenesis. Current studies are comparing p100 processing and proteasome activity in iMLL-ALL with competent and disrupted HMGB1-RelB interactions, as well as comparing the effectiveness of proteasome inhibition with bortezomib. Near-future studies will screen other high-risk ALL's for HMGB1-Rel activity and elaborate the downstream effects of complex binding on the MLL-ALL transcriptome to establish it as a rational target for drug development.