Reciprocal deregulation of NKX3.1 and AURKA axis in castration-resistant prostate cancer and NEPC models.

BACKGROUND: NKX3.1, a prostate-specific tumor suppressor, is either genomically lost or its protein levels are severely downregulated, which are invariably associated with poor prognosis in prostate cancer (PCa). Nevertheless, a clear disconnect exists between its mRNA and protein levels, indicating that its post-translational regulation may be critical in maintaining its protein levels. Similarly, AURKA is vastly overexpressed in all stages of prostate cancer (PCa), including castration-resistant PCa (CRPC) and neuroendocrine PCa (NEPC), although its transcripts are only increased in ~ 15% of cases, hinting at additional mechanisms of deregulation. Thus, identifying the upstream regulators that control AURKA and NKX3.1's levels and/or their downstream effectors offer an alternative route to inhibit AURKA and upregulate NKX3.1 in highly fatal CRPC and NEPC. AURKA and NKX3.1 have not linked to each other in any study to date. METHODS: A chemical genetic screen revealed NKX3.1 as a direct target of AURKA. AURKA-NKX3.1 cross-talk was analyzed using several biochemical techniques in CRPC and NEPC cells. RESULTS: We uncovered a reciprocal loop between AURKA and NKX3.1 in CRPC and NEPC cells. We observed that AURKA-mediated NKX3.1 downregulation is a major mechanism that drives CRPC pathogenesis and NEPC differentiation. AURKA phosphorylates NKX3.1 at three sites, which degrades it, but AURKA does not regulate NKX3.1 mRNA levels. NKX3.1 degradation drives highly aggressive oncogenic phenotypes in cells. NKX3.1 also degrades AURKA in a feedback loop. NKX3.1-AURKA loop thus upregulates AKT, ARv7 and Androgen Receptor (AR)-signaling in tandem promoting highly malignant phenotypes. Just as importantly, we observed that NKX3.1 overexpression fully abolished synaptophysin and enolase expression in NEPC cells, uncovering a strong negative relationship between NKX3.1 and neuroendocrine phenotypes, which was further confirmed be measuring neurite outgrowth. While WT-NKX3.1 inhibited neuronal differentiation, 3A-NKX3.1 expression obliterated it. CONCLUSIONS: NKX3.1 loss could be a major mechanism causing AURKA upregulation in CRPC and NEPC and vice versa. NKX3.1 genomic loss requires gene therapy, nonetheless, targeting AURKA provides a powerful tool to maintain NKX3.1 levels. Conversely, when NKX3.1 upregulation strategy using small molecules comes to fruition, AURKA inhibition should work synergistically due to the reciprocal loop in these highly aggressive incurable diseases.

Global Proteomic Analyses of STING-Positive and -Negative Macrophages Reveal STING and Non-STING Differentially Regulated Cellular and Molecular Pathways.

PURPOSE: Cyclic guanosine monophosphate-adenosine monophosphate and other bacterial-derived cyclic di-guanosine monophosphate or cyclic di-adenosine monophosphate trigger innate immune responses through binding to stimulator of interferon genes (STING). Thus in chronic infection, such as in periodontitis, immune cells can be exposed to bacterial DNA and/or cyclic dinucleotides, potentially activating STING to cause inflammation. Thus far the cyclic GMP-AMP synthase-STING- TANK-binding kinase 1 pathway has been well characterized but a global perspective of how the presence or lack of STING affect the proteome is lacking. The aim of this study is to identify macrophage proteins that are affected by STING. EXPERIMENTAL DESIGN: Proteins are extracted from a macrophage cell line harboring STING (RAW-Blue ISG) as well as a STING knockout (STING KO) cell line (RAW-Lucia ISG-KO-STING) and global proteomics analyses are performed. RESULTS: Proteins related to kinase and phosphatase signaling, spliceosome, terpenoid backbone biosynthesis, glycosylation, ubiquitination, and phagocytosis are affected by STING knock out. CONCLUSIONS AND CLINICAL RELEVANCE: STING pathway in macrophages is related to the regulation of several proteins that are known as potent biomarkers of various cancers and autoimmune diseases. Moreover, the relation between STING and phagocytosis is demonstrated for the first time. Further validation studies will help identify molecules and pathways that may function as diagnostic or therapeutic targets.

Dual FLT3/TOPK inhibitor with activity against FLT3-ITD secondary mutations potently inhibits acute myeloid leukemia cell lines.

AIM: Approximately 30% of acute myeloid leukemia (AML) patients carry FLT3 tyrosine kinase domain (TKD) mutations or internal tandem duplication (FLT3-ITD). Currently there is a paucity of compounds that are active against drug-resistant FLT3-ITD, which contains secondary mutations in the TKD, mainly at residues D835/F691. RESULTS: HSD1169, a novel compound, is active against FLT3-ITD (D835 or F691). HSD1169 is also active against T-LAK cell-originated protein kinase (TOPK), a collaborating kinase that is highly expressed in AML cell lines. HSD1169 was active against MV4-11 and Molm-14 (FLT3-ITD cell lines) but not NOMO-1 or HL60 (FLT3-WT cell lines). HSD1169 was also active against sorafenib-resistant Molm13-res cell line (containing FLT3-ITD/D835Y). CONCLUSION: HSD1169 or an analog could become a therapeutic agent for AML containing drug-resistant FLT3-ITD.

Proteomic analysis of RAW macrophages treated with cGAMP or c-di-GMP reveals differentially activated cellular pathways.

Global and quantitative analysis of the proteome help to reveal how host cells sense invading bacteria and respond to bacterial signaling molecules. Here, we performed label free quantitative proteomic analysis of RAW macrophages treated with host-derived cGAMP and bacterial-derived c-di-GMP, in an attempt to identify cellular pathways impacted by these dinucleotides and determine if the host responds differentially to these two cyclic dinucleotides. We identified a total of 3811 proteins of which abundances of 404 proteins in cGAMP and 236 proteins in c-di-GMP treated cells were significantly different compared to the control. Many of the proteins that were strongly and commonly upregulated, such as interferon-induced proteins 47, 202 and 204 (Ifi47, Ifi202, Ifi204), ubiquitin-activating enzyme E7 (Uba7), interferon-induced protein with tetratricopeptide repeats 1, 2 or 3 (Ifit1, Ifit2, Ifit3), ubiquitin-like protein ISG15 (ISG15), might be due to the fact that both dinucleotides promote the production of interferons, which induce the expression of many proteins. However, there were also other proteins that were differentially affected by cGAMP or c-di-GMP treatment, including probable ATP-dependent RNA helicase DHX58 (Dhx58), nuclear autoantigen Sp-100 (Sp100), MARCKS-related protein (Marcksl1) and antigen peptide transporter 2 (Tap2). This is probably due to the differential levels of IFNs produced by the dinucleotides or may indicate that non-STING activation might also contribute to the host's response to c-di-GMP and cGAMP. Interestingly Trex1, a nuclease that degrades DNA (an activator of cGAS to produce cGAMP), was upregulated (3.22 fold) upon cGAMP treatment, hinting at a possible feedback loop to regulate cGAMP synthesis. These results lay a foundation for future studies to better characterize and understand the complex c-di-GMP and cGAMP signaling network.