HMGB3: A Novel Regulator of Leukemia Proliferation

Acute Myeloid leukemia (AML) is the deadliest form of leukemia in both adults and children. Due to the genetic heterogeneity of the disease, current AML treatments are ineffective for many patients. Even patients who initially respond to therapy can relapse and become unresponsive to treatment. This highlights the need to identify new therapies that can target AML regardless of the initiating mutation. In pursuit of this goal, we identified the chromatin binding factor high mobility group box 3 (HMGB3) as a potential therapeutic target. HMGB3 is primarily expressed in the nucleus of hematopoietic stem cells (HSCs) and regulates the balance between immature and mature blood cells. Previous work in vitro has implied a role for HMGB3 in leukemia stem cells as well; however, the mechanism for HMGB3’s role in these processes remains to be determined. HMGB3 is assumed to function similarly to its homologue HMGB1 but this has yet to be demonstrated experimentally. Determining how HMGB3 functions at a molecular level is a critical first step towards developing it as a therapeutic target in leukemia.

In initial studies, we found that HMGB3 protein is markedly increased as compared to healthy hematopoietic stem and progenitor cells (HSPCs) in a variety of leukemia cell lines, AML patient samples, and mouse models of AML regardless of the subtype. shRNA knockdown (KD) of HMGB3 in the human AML cell lines Kasumi-1 and MOLM13 showed that decreased HMGB3 results in a reduced growth rate. Flow cytometry analysis showed no difference in cell death, but a significant decrease in proliferation in KD cells. Additionally, KD cells showed an increase in cells in G0/G1 and a decrease in S phase indicating arrest in the early stages of the cell cycle. Colony assays of control and KD cells showed no difference in colony number, although colonies from HMGB3 KD cells were notably smaller supporting our finding that loss of HMGB3 causes a decrease in leukemia cell proliferation. This data suggests that HMGB3 promotes leukemia cell growth in vitro. To identify potential mechanisms mediating this activity, we performed whole transcriptome sequencing of control and HMGB3 KD MOLM13 cells and identified 131 differentially expressed genes. Gene Ontology analysis showed that the downregulated genes were associated with regulation of the MAPK signaling pathway. By western blot, we found a reduction in phosphorylated p38/MAPK, ERK1/2, and MEK1/2 but not total abundance of the proteins, in HMGB3 KD cells. Taken together, these data indicate that loss of HMGB3 downregulates MAPK activation, providing a potential mechanism for HMGB3’s control of proliferation.

To determine how a primarily nuclear protein regulates a cytosolic signaling pathway, we utilized information from the related protein, HMGB1. Localization and function of HMGB1 is determined by the redox state of three cysteine residues which are conserved in HMGB3. To tested if these cysteines in HGB3 are also susceptible to redox regulation, we treated leukemia cells with the oxidizing agent diamide. We found that increasing concentrations of diamide resulted in a shift from a reduced to an oxidized form of HMGB3. To determine if redox modulation of the cysteine residues affects HMGB3 localization, we mutated the cysteines to serines or aspartic acids to mimic the reduced and oxidized forms of the protein, respectively. In contrast to the wildtype protein, which is localized to the nucleus, mutants mimicking a fully oxidized protein were entirely cytosolic, implying that like HMGB1, high levels of reactive oxygen species (ROS) could affect HMGB3 localization. Because leukemia cells are known to have increased ROS as compared to healthy cells, we compared HMGB3 localization in leukemia and HSPCs and found that in both a mouse leukemia model and in leukemia patient samples HMGB3 is found in distinct puncta in the cytoplasm rather than the nucleus. Furthermore, we found that HMGB3 is secreted from leukemia cells in vitro, suggesting that HMGB3 may be regulating MAPK signaling through its extracellular form. Collectively, our results indicated that HMGB3 is an important regulator of leukemia cell proliferation and that understanding the mechanism of its activity could have important implication for the development of more effective treatments for patients with leukemia.