Entinostat in patients with relapsed or refractory abdominal neuroendocrine tumors.

BACKGROUND: Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) are rare neoplasms with an increasing annual incidence and prevalence. Many are metastatic at presentation or recur following surgical resection and require systemic therapy, for which somatostatin analogs such as octreotide or lanreotide comprise typical first-line therapies. Nonetheless, treatment options remain limited. Epigenetic processes such as histone modifications have been implicated in malignant transformation and progression. In this study, we evaluated the anti-proliferative effects of a histone deacetylase (HDAC) inhibitor, entinostat, which was computationally predicted to show anti-cancer activity, as confirmed in in vitro and in vivo models of GEP-NETs. METHODS: This was a phase II study to evaluate the efficacy and safety of entinostat in patients with relapsed or refractory abdominal NETs. The primary objective was to estimate the objective response rate to entinostat. Additionally, with each patient as his/her own control we estimated the rates of tumor growth prior to enrollment on study and while receiving entinostat. Patients received 5 mg entinostat weekly until disease progression or intolerable toxicity. The dose could be changed to 10 mg biweekly for patients who did not experience grade ≥ 2 treatment-related adverse events (AEs) in cycle 1, but was primarily administered at the starting 5 mg weekly dose. RESULTS: The study enrolled only 5 patients due to early termination by the drug sponsor. The first patient that enrolled had advanced disease and died within days of enrollment before follow-up imaging due to a grade 5 AE unrelated to study treatment and was considered non-evaluable. Best RECIST response for the remaining 4 patients was stable disease (SD) with time on study of 154+, 243, 574, and 741 days. With each patient as his/her own control, rates of tumor growth on entinostat were markedly reduced with rates 20%, 33%, 54%, and 68% of the rates prior to enrollment on study. Toxicities possibly or definitely related to entinostat included grade 2/3 neutrophil count decrease [2/4 (50%)/ 2/4 (50%)], grade 3 hypophosphatemia [1/4, (25%)], grade 1/2 fatigue [1/4 (25%)/ 2/4 (50%)], and other self-limiting grade 1/2 AEs. CONCLUSION: In the treatment of relapsed or refractory abdominal NETs, entinostat 5 mg weekly led to prolonged SD and reduced the rate of tumor growth by 32% to 80% with an acceptable safety profile (ClinicalTrials.gov Identifier: NCT03211988).

Androgen-targeting therapeutics mitigate the adverse effect of GnRH agonist on the risk of neurodegenerative disease in men treated for prostate cancer.

BACKGROUND: Prostate cancer and multiple neurodegenerative diseases (NDD) share an age-associated pattern of onset. Therapy of prostate cancer is known to impact cognitive function. The objective of this study was to determine the impact of multiple classes of androgen-targeting therapeutics (ATT) on the risk of NDD. METHODS: A retrospective cohort study of men aged 45 and older with prostate within the US-based Mariner claims data set between January 1 and 27, 2021. A propensity score approach was used to minimize measured and unmeasured selection bias. Disease risk was determined using Kaplan-Meier survival analyses. RESULTS: Of the 1,798,648 men with prostate cancer, 209,722 met inclusion criteria. Mean (SD) follow-up was 6.4 (1.8) years. In the propensity score-matched population, exposure to ATT was associated with a minimal increase in NDD incidence (relative risk [RR], 1.07; 95% CI, 1.05-1.10; p < 0.001). However, GnRH agonists alone were associated with significantly increased NDD risk (RR, 1.47; 95% CI, 1.30-1.66; p <0.001). Abiraterone, commonly administered with GnRH agonists and low-dose prednisone, was associated with a significantly decreased risk (RR, 0.77; 95% CI, 0.68-0.87; p < 0.001) of any NDD. CONCLUSIONS: Among patients with prostate cancer, GnRH agonist exposure was associated with an increased NDD risk. Abiraterone acetate reduced the risks of Alzheimer's disease and Parkinson's disease conferred by GnRH agonists, whereas the risk for ALS was reduced by androgen receptor inhibitors. Outcomes of these analyses contribute to addressing controversies in the field and indicate that GnRH agonism may be a predictable instigator of risk for NDD with opportunities for risk mitigation in combination with another ATT.

CRISPR/Cas9-Mediated Point Mutation in Nkx3.1 Prolongs Protein Half-Life and Reverses Effects Nkx3.1 Allelic Loss.

NKX3.1 is the most commonly deleted gene in prostate cancer and is a gatekeeper suppressor. NKX3.1 is haploinsufficient, and pathogenic reduction in protein levels may result from genetic loss, decreased transcription, and increased protein degradation caused by inflammation or PTEN loss. NKX3.1 acts by retarding proliferation, activating antioxidants, and enhancing DNA repair. DYRK1B-mediated phosphorylation at serine 185 of NKX3.1 leads to its polyubiquitination and proteasomal degradation. Because NKX3.1 protein levels are reduced, but never entirely lost, in prostate adenocarcinoma, enhancement of NKX3.1 protein levels represents a potential therapeutic strategy. As a proof of principle, we used CRISPR/Cas9-mediated editing to engineer in vivo a point mutation in murine Nkx3.1 to code for a serine to alanine missense at amino acid 186, the target for Dyrk1b phosphorylation. Nkx3.1S186A/-, Nkx3.1+/- , and Nkx3.1+/+ mice were analyzed over one year to determine the levels of Nkx3.1 expression and effects of the mutant protein on the prostate. Allelic loss of Nkx3.1 caused reduced levels of Nkx3.1 protein, increased proliferation, and prostate hyperplasia and dysplasia, whereas Nkx3.1S186A/- mouse prostates had increased levels of Nkx3.1 protein, reduced prostate size, normal histology, reduced proliferation, and increased DNA end labeling. At 2 months of age, when all mice had normal prostate histology, Nkx3.1+/- mice demonstrated indices of metabolic activation, DNA damage response, and stress response. These data suggest that modulation of Nkx3.1 levels alone can exert long-term control over premalignant changes and susceptibility to DNA damage in the prostate. SIGNIFICANCE: These findings show that prolonging the half-life of Nkx3.1 reduces proliferation, enhances DNA end-labeling, and protects from DNA damage, ultimately blocking the proneoplastic effects of Nkx3.1 allelic loss.

Loss of PTEN Accelerates NKX3.1 Degradation to Promote Prostate Cancer Progression.

NKX3.1 is the most commonly deleted gene in prostate cancer and a gatekeeper suppressor. NKX3.1 is a growth suppressor, mediator of apoptosis, inducer of antioxidants, and enhancer of DNA repair. PTEN is a ubiquitous tumor suppressor that is often decreased in prostate cancer during tumor progression. Steady-state turnover of NKX3.1 is mediated by DYRK1B phosphorylation at NKX3.1 serine 185 that leads to polyubiquitination and proteasomal degradation. In this study, we show PTEN is an NKX3.1 phosphatase that protects NKX3.1 from degradation. PTEN specifically opposed phosphorylation at NKX3.1(S185) and prolonged NKX3.1 half-life. PTEN and NKX3.1 interacted primarily in the nucleus as loss of PTEN nuclear localization abrogated its ability to bind to and protect NKX3.1 from degradation. The effect of PTEN on NKX3.1 was mediated via rapid enzyme-substrate interaction. An effect of PTEN on Nkx3.1 gene transcription was seen in vitro, but not in vivo. In gene-targeted mice, Nkx3.1 expression significantly diminished shortly after loss of Pten expression in the prostate. Nkx3.1 loss primarily increased prostate epithelial cell proliferation in vivo. In these mice, Nkx3.1 mRNA was not affected by Pten expression. Thus, the prostate cancer suppressors PTEN and NKX3.1 interact and loss of PTEN is responsible, at least in part, for progressive loss of NKX3.1 that occurs during tumor progression. SIGNIFICANCE: PTEN functions as a phosphatase of NKX3.1, a gatekeeper suppressor of prostate cancer.

Tissue microarray analysis delineate potential prognostic role of Annexin A7 in prostate cancer progression.

BACKGROUND: Annexin A7 (ANXA7) is a member of the multifunctional calcium or phospholipid-binding annexin gene family. While low levels of ANXA7 are associated with aggressive types of cancer, the clinical impact of ANXA7 in prostate cancer remains unclear. Tissue microarrays (TMA) have revealed several new molecular markers in human tumors. Herein, we have identified the prognostic impact of ANXA7 in a prostate cancer using a tissue microarray containing 637 different specimens. METHODS: The patients were diagnosed with prostate cancer and long-term follow-up information on progression (median 5.3 years), tumor-specific and overall survival data (median 5.9 years) were available. Expression of Ki67, Bcl-2, p53, CD-10 (neutral endopeptidase), syndecan-1 (CD-138) and ANXA7 were analyzed by immunohistochemistry. RESULTS: A bimodal distribution of ANXA7 was observed. Tumors expressing either high or no ANXA7 were found to be associated with poor prognosis. However, ANXA7 at an optimal level, in between high and no ANXA7 expression, had a better prognosis. This correlated with low Ki67, Bcl-2, p53 and high syndecan-1 which are known predictors of early recurrence. At Gleason grade 3, ANXA7 is an independent predictor of poor overall survival with a p-value of 0.003. Neoadjuvant hormonal therapy, which is known to be associated with overexpression of Bcl-2 and inhibition of Ki67 LI and CD-10, was found to be associated with under-expression of ANXA7. CONCLUSIONS: The results of this TMA study identified ANXA7 as a new prognostic factor and indicates a bimodal correlation to tumor progression.

Development, Evaluation, and Implementation of a Pan-African Cancer Research Network: Men of African Descent and Carcinoma of the Prostate.

PURPOSE: Cancer of the prostate (CaP) is the leading cancer among men in sub-Saharan Africa (SSA). A substantial proportion of these men with CaP are diagnosed at late (usually incurable) stages, yet little is known about the etiology of CaP in SSA. METHODS: We established the Men of African Descent and Carcinoma of the Prostate Network, which includes seven SSA centers partnering with five US centers to study the genetics and epidemiology of CaP in SSA. We developed common data elements and instruments, regulatory infrastructure, and biosample collection, processing, and shipping protocols. We tested this infrastructure by collecting epidemiologic, medical record, and genomic data from a total of 311 patients with CaP and 218 matched controls recruited at the seven SSA centers. We extracted genomic DNA from whole blood, buffy coat, or buccal swabs from 265 participants and shipped it to the Center for Inherited Disease Research (Baltimore, MD) and the Centre for Proteomics and Genomics Research (Cape Town, South Africa), where genotypes were generated using the UK Biobank Axiom Array. RESULTS: We used common instruments for data collection and entered data into the shared database. Double-entered data from pilot participants showed a 95% to 98% concordance rate, suggesting that data can be collected, entered, and stored with a high degree of accuracy. Genotypes were obtained from 95% of tested DNA samples (100% from blood-derived DNA samples) with high concordance across laboratories. CONCLUSION: We provide approaches that can produce high-quality epidemiologic and genomic data in multicenter studies of cancer in SSA.

Nkx3.1 controls the DNA repair response in the mouse prostate.

BACKGROUND: The human prostate tumor suppressor NKX3.1 mediates the DNA repair response and interacts with the androgen receptor to assure faithful completion of transcription thereby protecting against TMPRSS2-ERG gene fusion. To determine directly the effect of Nkx3.1 in vivo we studied the DNA repair response in prostates of mice with targeted deletion of Nkx3.1. METHODS: Using both drug-induced DNA damage and γ-irradiation, we assayed expression of γ-histone 2AX at time points up to 24 hr after induction of DNA damage. RESULTS: We demonstrated that expression of Nkx3.1 influenced both the timing and magnitude of the DNA damage response in the prostate. CONCLUSIONS: Nkx3.1 affects the DNA damage response in the murine prostate and is haploinsufficient for this phenotype.

NKX3.1 Suppresses TMPRSS2-ERG Gene Rearrangement and Mediates Repair of Androgen Receptor-Induced DNA Damage.

TMPRSS2 gene rearrangements occur at DNA breaks formed during androgen receptor-mediated transcription and activate expression of ETS transcription factors at the early stages of more than half of prostate cancers. NKX3.1, a prostate tumor suppressor that accelerates the DNA repair response, binds to androgen receptor at the ERG gene breakpoint and inhibits both the juxtaposition of the TMPRSS2 and ERG gene loci and also their recombination. NKX3.1 acts by accelerating DNA repair after androgen-induced transcriptional activation. NKX3.1 influences the recruitment of proteins that promote homology-directed DNA repair. Loss of NKX3.1 favors recruitment to the ERG gene breakpoint of proteins that promote error-prone nonhomologous end-joining. Analysis of prostate cancer tissues showed that the presence of a TMPRSS2-ERG rearrangement was highly correlated with lower levels of NKX3.1 expression consistent with the role of NKX3.1 as a suppressor of the pathogenic gene rearrangement.

The Tumor Suppressor NKX3.1 Is Targeted for Degradation by DYRK1B Kinase.

UNLABELLED: NKX3.1 is a prostate-specific homeodomain protein and tumor suppressor whose expression is reduced in the earliest phases of prostatic neoplasia. NKX3.1 expression is not only diminished by genetic loss and methylation, but the protein itself is a target for accelerated degradation caused by inflammation that is common in the aging prostate gland. NKX3.1 degradation is activated by phosphorylation at C-terminal serine residues that mediate ubiquitination and protein turnover. Because NKX3.1 is haploinsufficient, strategies to increase its protein stability could lead to new therapies. Here, a high-throughput screen was developed using an siRNA library for kinases that mediate NKX3.1 degradation. This approach identified several candidates, of which DYRK1B, a kinase that is subject to gene amplification and overexpression in other cancers, had the greatest impact on NKX3.1 half-life. Mechanistically, NKX3.1 and DYRK1B were shown to interact via the DYRK1B kinase domain. In addition, an in vitro kinase assay showed that DYRK1B phosphorylated NKX3.1 at serine 185, a residue critical for NKX3.1 steady-state turnover. Lastly, small-molecule inhibitors of DYRK1B prolonged NKX3.1 half-life. Thus, DYRK1B is a target for enzymatic inhibition in order to increase cellular NKX3.1. IMPLICATIONS: DYRK1B is a promising and novel kinase target for prostate cancer treatment mediated by enhancing NKX3.1 levels.

Functional activation of ATM by the prostate cancer suppressor NKX3.1.

The prostate tumor suppressor NKX3.1 augments response to DNA damage and enhances survival after DNA damage. Within minutes of DNA damage, NKX3.1 undergoes phosphorylation at tyrosine 222, which is required for a functional interaction with ataxia telangiectasia mutated (ATM) kinase. NKX3.1 binds to the N-terminal region of ATM, accelerates ATM activation, and hastens the formation of γhistone2AX. NKX3.1 enhances DNA-dependent ATM kinase activation by both the MRN complex and H2O2 in a DNA-damage-independent manner. ATM, bound to the NKX3.1 homeodomain, phosphorylates NKX3.1, leading to ubiquitination and degradation. Thus, NKX3.1 and ATM have a functional interaction leading to ATM activation and then NKX3.1 degradation in a tightly regulated DNA damage response specific to prostate epithelial cells. These findings demonstrate a mechanism for the tumor-suppressor properties of NKX3.1, demonstrate how NKX3.1 may enhance DNA integrity in prostate stem cells and may help to explain how cells differ in their sensitivity to DNA damage.

Structural and functional interactions of the prostate cancer suppressor protein NKX3.1 with topoisomerase I.

NKX3.1 (NK3 homeobox 1) is a prostate tumour suppressor protein with a number of activities that are critical for its role in tumour suppression. NKX3.1 mediates the cellular response to DNA damage by interacting with ATM (ataxia telangiectasia mutated) and by activation of topoisomerase I. In the present study we characterized the interaction between NKX3.1 and topoisomerase I. The NKX3.1 homeodomain binds to a region of topoisomerase I spanning the junction between the core and linker domains. Loss of the topoisomerase I N-terminal domain, a region for frequent protein interactions, did not affect binding to NKX3.1 as was shown by the activation of Topo70 (N-terminal truncated topoisomerase I) in vitro. In contrast, NKX3.1 interacts with the enzyme reconstituted from peptide fragments of the core and linker active site domains, but inhibits the DNA-resolving activity of the reconstituted enzyme in vitro. The effect of NKX3.1 on both Topo70 and the reconstituted enzyme was seen in the presence and absence of camptothecin. Neither NKX3.1 nor CPT (camptothecin) had an effect on the interaction of the other with topoisomerase I. Therefore the interactions of NKX3.1 and CPT with the linker domain of topoisomerase I are mutually exclusive. However, in cells the effect of NKX3.1 on topoisomerase binding to DNA sensitized the cells to cellular toxicity and the induction of apoptosis by low doses of CPT. Lastly, topoisomerase I is important for the effect of NKX3.1 on cell survival after DNA damage as topoisomerase knockdown blocked the effect of NKX3.1 on clonogenicity after DNA damage. Therefore NKX3.1 and topoisomerase I interact in vitro and in cells to affect the CPT sensitivity and DNA-repair functions of NKX3.1.

Renal cell cancer treated with a single-edged sword.

Preclinical and early clinical data suggest that antiangiogenic treatments may lead to more aggressive tumors. In this issue of Cell Reports, Blagoev et al. (2013) show that sunitinib, a multikinase inhibitor with antiangiogenic effects, does not worsen the survival of patients with metastatic kidney cancer.

Variant NKX3.1 and Serum IGF-1: Investigation of Interaction in Prostate Cancer.

NKX3.1 is a tumor suppressor down-regulated in early prostate cancers. A SNP (rs2228013), which represents a polymorphic NKX3.1(C154T) coding for a variant protein NKX3.1(R52C), is present in 10% of the population and is related to prostatic enlargement and prostate cancer. We investigated rs2228013 in prostate cancer risk for 937 prostate cancer cases and 1,086 age-matched controls from a nested case-control study within the prospective Physicians' Health Study (PHS) and among 798 cases and 527 controls retrospectively collected in the Risk Factors for Prostate Cancer Study of the Victoria Cancer Council (RFPCS). We also investigated the interaction between serum IGF-I levels and NKX3.1 genotype in the populations from PHS and RFPCS. In the PHS, we found no overall association between the variant T allele in rs2228013 in NKX3.1 and prostate cancer risk (odd ratio = 1.25; 95% confidence interval = 0.92-1.71). A subgroup analysis for cases diagnosed before age 70 showed an increased risk (relative risk = 1.55; 95% confidence interval = 1.04-2.31) of overall prostate cancer. In this age-group, the risk of metastatic cancer at diagnosis or of fatal cancer was even higher in carriers of the T allele (relative risk = 2.15; 95% confidence interval = 1.00-4.63). These associations were not replicated in the RFPCS. Serum IGF-I levels were found to be a risk factor for prostate cancer in both study populations. The wild type NKX3.1 protein can induce IGFBP-3 expression in vitro. We report that variant NKX3.1 cannot induce IGFBP-3 expression, but the NKX3.1 genotype does not modify the association between serum IGF-I levels and prostate cancer risk.

High NRBP1 expression in prostate cancer is linked with poor clinical outcomes and increased cancer cell growth.

BACKGROUND: We recently established the rationale that NRBP1 (nuclear receptor binding protein 1) has a potential growth-promoting role in cell biology. NRBP1 interacts directly with TSC-22, a potential tumor suppressor gene that is differently expressed in prostate cancer. Consequently, we analyzed the role of NRBP1 expression in prostate cancer cell lines and its expression on prostate cancer tissue microarrays (TMA). METHODS: The effect of NRBP1 expression on tumor cell growth was analyzed by using RNAi. NRBP1 protein expression was evaluated on two TMAs containing prostate samples from more than 1,000 patients. Associations with clinico-pathological features, the proliferation marker Ki67 and survival data were analyzed. RESULTS: RNAi mediated silencing of NRBP1 expression in prostate cancer cell lines resulted in reduced cell growth (P < 0.05). TMA analysis revealed NRBP1 protein expression in benign prostate hyperplasia in 6% as compared to 60% in both, high-grade intraepithelial neoplasia and prostate cancer samples. Strong NRBP1 protein expression was restricted to prostate cancer and correlated with higher expression of the proliferation marker Ki67 (P < 0.05). Further, patients with strong NRBP1 protein expression showed poor clinical outcomes (P < 0.05). Analysis of matched localized cancer tissues before and after castration revealed that post-therapy-related repression of NRBP1 expression was significantly associated with better overall survival. CONCLUSIONS: We demonstrate that expression of NRBP1 is up-regulated during the progression of prostate cancer and that high NRBP1 expression is linked with poor prognosis and enhanced tumor cell growth.

NKX3.1 activates cellular response to DNA damage.

The prostate-specific tumor suppressor homeodomain protein NKX3.1 is inactivated by a variety of mechanisms in the earliest phases of prostate carcinogenesis and in premalignant regions of the prostate gland. The mechanisms by which NKX3.1 exercises tumor suppression have not been well elucidated. Here, we show that NKX3.1 affects DNA damage response and cell survival after DNA damage. NKX3.1 expression in PC-3 prostate cancer cells enhances colony formation after DNA damage but has minimal effect on apoptosis. NKX3.1 also diminishes and regulates total cellular accumulation of gammaH2AX. Endogenous NKX3.1 in LNCaP cells localizes to sites of DNA damage where it affects the recruitment of phosphorylated ATM and the phosphorylation of H2AX. Knockdown of NKX3.1 in LNCaP cells attenuates the acute responses of both ATM and H2AX phosphorylation to DNA damage and their subnuclear localization to DNA damage sites. NKX3.1 expression enhances activation of ATM as assayed by autophosphorylation at serine 1981 and activation of ATR as assayed by phosphorylation of CHK1. An inherited mutation of NKX3.1 that predisposes to early prostate cancer and attenuates in vitro DNA binding was devoid of the ability to activate ATM and to colocalize with gammaH2AX at foci of DNA damage. These data show a novel mechanism by which a homeoprotein can affect DNA damage repair and act as a tumor suppressor.

Cross-regulation of signaling pathways: an example of nuclear hormone receptors and the canonical Wnt pathway.

Predicting the potential physiological outcome(s) of any given molecular pathway is complex because of cross-talk with other pathways. This is particularly evident in the case of the nuclear hormone receptor and canonical Wnt pathways, which regulate cell growth and proliferation, differentiation, apoptosis, and metastatic potential in numerous tissues. These pathways are known to intersect at many levels: in the intracellular space, at the membrane, in the cytoplasm, and within the nucleus. The outcomes of these interactions are important in the control of stem cell differentiation and maintenance, feedback loops, and regulating oncogenic potential. The aim of this review is to demonstrate the importance of considering pathway cross-talk when predicting functional outcomes of signaling, using nuclear hormone receptor/canonical Wnt pathway cross-talk as an example.

Primary vulvar Ewing sarcoma/primitive neuroectodermal tumor: a report of 2 cases and review of the literature.

Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) family of tumor is a very aggressive malignant round cell tumor characterized by translocations involving EWS-FLI1 genes. They are increasingly recognized in extraosseous sites as a result of improvements in diagnostic tools. In this paper, we report 2 additional cases arising in vulva of young adults who have been treated aggressively and have survived fore more than 7 and 4 years successively. Histologic examination showed small round (blue) cell morphology in both cases. The tumor cells contained glycogen and were positive for CD99 and vimentin and negative for keratins, lymphoid markers, S-100, synaptophysin, chromogranin, and desmin. Reverse transcriptase polymerase chain reaction analysis from paraffin-embedded tissue revealed EWS-FLI1 fusion product in 1 case. Collectively, 13 cases of vulvar ES/PNET have been reported in the literature. Only 8 cases have detailed follow-up information with an average follow-up data of 28 months. Ewing sarcoma/PNET should be considered in the differential diagnosis of any undifferentiated tumors involving the lower gynecologic tract and all axillary tests including molecular tests should be performed for correct diagnosis because prolonged survival is possible for this dreadful disease after complete surgical resection, followed by adjuvant therapy.

Interactions of the acidic domain and SRF interacting motifs with the NKX3.1 homeodomain.

NKX3.1 is a prostate tumor suppressor belonging to the NK-2 family of homeodomain (HD) transcription factors. NK-2 family members often possess a stretch of 10-15 residues enriched in acidic amino acids, the acidic domain (AD), in the flexible, disordered region N-terminal to the HD. Interactions between the N-terminal region of NKX3.1 and its homeodomain affect protein stability and DNA binding. CD spectroscopy measuring the thermal unfolding of NKX3.1 constructs showed a 2 degrees C intramolecular stabilization of the HD by the N-terminal region containing the acidic domain (residues 85-96). CD of mixtures of various N-terminal peptides with a construct containing just the HD showed that the acidic domain and the following region, the SRF interacting (SI) motif (residues 99-105), was necessary for this stabilization. Phosphorylation of the acidic domain is known to slow proteasomal degradation of NKX3.1 in prostate cells, and NMR spectroscopy was used to measure and map the interaction of the HD with phosphorylated and nonphosphorylated forms of the AD peptide. The interaction with the phosphorylated AD peptide was considerably stronger (K(d) = 0.5 +/- 0.2 mM), resulting in large chemical shift perturbations for residues Ser150 and Arg175 in the HD, as well as a 2 degrees C increase in the HD thermal stability compared to that of the nonphosphorylated form. NKX3.1 constructs with AD phosphorylation site threonine residues (89 and 93) mutated to glutamate were 4 degrees C more stable than HD alone. Using polymer theory, effective concentrations for interactions between domains connected by flexible linkers are predicted to be in the millimolar range, and thus, the weak intramolecular interactions observed here could conceivably modulate or compete with stronger, intermolecular interactions with the NKX3.1 HD.