ALBERT

Description: Elevated plasma levels of the nucleoside diphosphate kinase (NDPK) NM23-H1 are associated with poorer prognosis in acute myeloid leukemia (AML). We previously demonstrated that leukemic blasts release NM23-H1, which binds to more differentiated myeloid cells inducing their secretion of inflammatory cytokines, including IL-1β, that promote survival and proliferation of leukemic blasts1. Both AML and myelodysplastic syndrome (MDS) patients are prone to infections due to impaired hematopoiesis that is worsened by treatment. NDPKs are highly evolutionarily conserved raising the possibility that bacterial/fungal NDPKs could mediate the same survival effect on malignant AML/MDS blasts and exacerbate disease progression. To test this, we generated recombinant NDPKs (rNDPKs) from bacteria and fungi associated with common infections in these patients (E. coli, S. aureus, S. pneumoniae, K. pneumoniae, C. albicans). Cytokine production and survival responses of primary AMLs to these proteins were indistinguishable from their response to rNM23-H1. This activity was independent of NDPK enzyme activity since mutant rNM23-H1 and bacterial and fungal rNDPKs with impaired oligomerization, kinase or exonuclease activity elicited the same cytokine and survival response. Toll like receptors (TLRs) are the major family of human DAMP/PAMP receptors and IL-1β secretion is closely associated with TLR-4 mediated activation of the NLRP3 inflammasome in monocytes. We therefore postulated that NM23-H1 and pathogen derived NDPKs act as novel damage- and pathogen- associated molecular pattern (DAMP, PAMP) molecules. We confirmed that fluorescently labelled rNM23-H1 and S. pneumoniae rNDPK bound selectively to monocytes in peripheral blood. Using in vitro generated monocytes (vitamin D3 differentiated THP-1 cells) we demonstrated that both wild type and mutant rNM23-H1 and bacterial/fungal rNDPKs induced activation of caspase-1 and cleavage of pro-IL-1β into its active form. Secretion of IL-1β was inhibited by antagonists/inhibitors of TLR4, NLRP3 and caspase-1 indicating the involvement of the TLR4-NLRP3 inflammasome axis is mediating the NDPK response. Unlike the canonical NLRP3-inflammasome pathway that leads to monocyte cell death by pyroptosis, rNM23-H1 and rNDPKs did not lead to cell death indicating that rNDPKs are responsible for the activation of the alternative inflammasome. In our earlier studies, and those of others, we demonstrated that not all AML primary samples responsed to NM23-H1 in vitro. We have observed that non responders to NM23-H1 also do not respond to pathogen derived rNDPKs. In contrast, we have observed uniform responses in terms of cytokine release in all normal peripheral blood. We hence hypothesized that the non-rNDPK-responding AML samples may reflect the absence of monocytes in culture. To test this, we generated conditioned media using normal donor leukocytes, in presence or absence of a TLR-4 antagonist to inhibit the IL-1β production. The conditioned media was then used to culture primary AML samples, in parallel with rNDPK in unconditioned media. All the samples analyzed showed increased survival in rNDPK conditioned media even whilst some did not respond to rNDPK in unconditioned media. In summary, our data demonstrate for the first time that NM23-H1 and bacterial/fungal NDPKs are novel DAMPs/PAMPS that signal via TLR4 in monocytes. We further demonstrate that this interaction results in activation of the alternative NLRP3 inflammasome and subsequent cleavage and secretion of IL-1β without death by pyroptosis. Our data showing that bacterial/fungal NDPKs can promote survival of AML blasts indicates that rather than just being a consequence of AML associated immunosuppression, infections may drive the progression and AML. These findings have important implications in the clinical management of AML and its precursor myelodysplastic syndromes (MDS). Lilly AJ, et al.Cancer Res. 2011;71(3):1177-86.Gaidt MM, et al. Immunity. 2016;44(4):833-46 Disclosures Drayson: Abingdon Health: Consultancy, Equity Ownership.

Description: Some highly metastatic types of breast cancer show decreased intracellular levels of the tumor suppressor protein NME1, also known as nm23-H1 or nucleoside diphosphate kinase A (NDPK-A), which decreases cancer cell motility and metastasis. Since its activity is directly correlated with the overall outcome in patients, increasing the cytosolic levels of NDPK-A/NME1 in such cancer cells should represent an attractive starting point for novel therapeutic approaches to reduce tumor cell motility and decrease metastasis. Here, we established the Bacillus anthracis protein toxins’ transport component PA63 as transporter for the delivery of His-tagged human NDPK-A into the cytosol of cultured cells including human MDA-MB-231 breast cancer cells. The specifically delivered His6-tagged NDPK-A was detected in MDA-MB-231 cells via Western blotting and immunofluorescence microscopy. The PA63-mediated delivery of His6-NDPK-A resulted in reduced migration of MDA-MB-231 cells, as determined by a wound-healing assay. In conclusion, PA63 serves for the transport of the tumor metastasis suppressor NDPK-A/NME1 into the cytosol of human breast cancer cells In Vitro, which reduced the migratory activity of these cells. This approach might lead to development of novel therapeutic options.

Description: Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.