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Somatostatin (somatotropin release inhibiting factor) is an abundant neuropeptide, which acts on five subtypes of somatostatin receptor (SST1-SST5; nomenclature as agreed by the NC-IUPHAR Subcommittee on Somatostatin Receptors [98]). Activation of these receptors produces a wide range of physiological effects throughout the body including the inhibition of secretion of many hormones. Endogenous ligands for these receptors are somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28). cortistatin-14 has also been suggested to be an endogenous ligand for somatostatin receptors [61].

Supplementary Table 1: Table provides information on the siRNA sequences and/or chemical structures of the non-FDA approved inhibitors for bromodomain proteins, transcription factors and kinases described in the current study. Supplementary Table 2: Sequences of primers used for qRT-PCR analysis of various targets described in the study. Supplementary Table 3: Sequences of primers used for ChIP analysis of various targets described in the study. Supplementary Table 4: Analysis of HOXB13 target gene expression in the GSE92721 data set. Supplementary Table 5: Analysis of HOTBIN10 gene expression in the GSE92721data set. Supplementary Table 6: Analysis of HOTBIN10 gene expression in the Moffitt TCC data set. Supplementary Table 7: Clinical parameters of the primary cancers in the Moffitt TCC data set. Supplementary Table 8: Clinical parameters of the metastatic cancers in the Moffitt TCC data set. Supplementary Table 9: Analysis of HOTBIN10 genes in MSKCC gene expression data set (GSE21034). Supplementary Table 10: Analysis of HOTBIN10 gene expression in the GSE67980 CTC data set.

Supplementary Fig. 1. Epigenetic landscape and the transcription cofactor recruitment at the HOXB13 enhancer region under various treatments; Supplementary Figure 2. BRD4 is a target of the dual BET-kinase inhibitors in CRPCs; Supplementary Figure 3. Sensitivity of normal and AR negative cell lines to the novel BET-kinase inhibitors; Supplementary Figure 4. HOXB13 but not FOXA1 is under the epigenetic control of BRD4 in prostate cancer cells; Supplementary Figure S5. Haploinsufficiency of HOXB13 sensitizes CRPCs to the novel BET-kinase inhibitors; Supplementary Figure S6. AURKB expression correlates with HOXB13 in human CTCs from mCRPC patients following Abiraterone therapy; Supplementary Fig. S7. AURKB is a therapeutic vulnerability of HOXB13 positive prostate cancers

Multiple myeloma (MM) is an incurable disease. Classical chemotherapeutics including bortezomib, melphalan, lenalidomide and thalidomide have greatly enhanced survival times. Unfortunately, patients typically relapse and become refractory with an average survival of 5 years post-diagnosis. Our emerging studies demonstrate a novel role for ULK3 in regulating autophagy in MM, a key program that sustains cell survival under times of stress and has been implicated as a major mechanism of proteasome inhibitor (PI) resistance. MM is known to be highly dependent on autophagy and, currently, specific ULK3 inhibitors are lacking. We posit that by targeting this marker in chemotherapy resistant MM patients, we can circumvent alternative metabolic routes and resensitize to standard of care proapoptotic therapy. We performed RNASeq analysis of CD138+ MM cells derived from patients across the disease stages spectrum (n=815) to confirm the role of ULK3 in disease progression and resistance to chemotherapy. We developed novel inhibitors SG3014/MA9060 that target multiple kinases including ULK3 (EC50 90nM) as well as BRD4. BRD4 is a known driver of MYC and its expression is increased in refractory MM. The BRD4 inhibitor, JQ1, effectively impairs the tumorigenic potential of MM but resistance has also been noted. We determined the efficacy of MA9060 for the treatment of CD138+ MM isolated from naive and refractory patients using a novel ex vivo high throughput platform developed at Moffitt.ULK3 is highly associated with MM stage of the disease. Refractory MM patients have increased autophagy activity with significantly higher expression of ULK3 in refractory patients and in drug resistant cell lines (immunoblotting U266 vs U266-PSR; RPMI-8226 vs RPMI-8226-B25; ABNL vs V10 resistant cells).Genetic ablation of ULK3 by siRNA in U266 and 8226 cell lines results in rapid cessation of the downstream autophagy proteins (ULK1, ATG13, pATG13) and MM cell death within 72h of transduction. Increased concentrations of autophagy inhibitors MA9060/SG3014 progressively decreased CMYC and ULK3 levels, as measured by immunoblotting in U266 cells. In vivo preclinical model of U266Luc tail vein injection proved our drugs are highly effective in reducing tumor dissemination and extending overall survival (CTRL untreated n=65 days vs MA9060 n=110). Importantly, we noted no overt toxicity and protected effect against myeloma-induced bone disease. This novel class of drug works synergistically with PI and can re-sensitize PI resistant disease to these effective therapies. We also show by EMMA ex vivo platform that MA9060 is highly effective for the treatment of CD138+ MM cells isolated from patients with refractory disease.ULK3 represents a novel target for treatment of MM refractory disease. Our dual inhibitors can increase overall survival in vivo and ex vivo, therefore we expect to quickly translate our novel molecules to the clinic. Citation Format: Marilena Tauro, Tao Li, Mark Meads, Praneeth R. Sudalagunta, Raghunandan R. Alugubelli, Nicholas J. Lawrence, Ernst Schonbrunn, Harshani Lawrence, Kenneth H. Shain, Conor C. Lynch. Novel autophagy inhibitory strategies to overcome chemotherapy resistance in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 452.

Estimating the actual extent of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is challenging because virus test positivity data undercount the actual number and proportion of persons infected. SARS-CoV-2 seroprevalence is a marker of past SARS-CoV-2 infection regardless of presence or severity of symptoms and therefore is a robust biomarker of infection period prevalence. We estimated SARS-CoV-2 seroprevalence among residents of Hillsborough County, Florida, USA, to determine factors independently associated with SARS-CoV-2 antibody status overall and among asymptomatic antibody-positive persons. Among 867 participants, SARS-CoV-2 period prevalence (October 2020-March 2021) was 19.5% (asymptomatic seroprevalence was 8%). Seroprevalence was 2-fold higher than reported SARS-CoV-2 virus test positivity. Factors related to social distancing (e.g., essential worker status, not practicing social distancing, contact with a virus-positive person, and length of contact exposure time) were consistently associated with seroprevalence but did not differ by time since suspected or known infection (6 months vs.6 months).

Gene expression is regulated by promoters and enhancers marked by histone H3 lysine 27 acetylation (H3K27ac), which is established by the paralogous, multidomain-containing histone acetyltransferases (HAT) EP300 and CBP. These enzymes display overlapping regulatory roles in untransformed cells, but less characterized roles in cancer cells. We demonstrate that the majority of high-risk pediatric neuroblastoma (NB) depends on EP300, whereas CBP has a limited role. To disrupt EP300, we developed a proteolysis-targeting chimera (PROTAC) compound termed “JQAD1” that selectively targets EP300 for degradation. JQAD1 treatment causes loss of H3K27ac at the enhancers that regulate NB master transcription factors, and drives rapid NB apoptosis, with limited toxicity to untransformed cells where CBP may compensate. In parallel, we demonstrate that EP300 and CBP have subdomain-specific functions, with enriched activity for individual domains in distinct tumor lineages. These data provide a foundation for interrogation of these histone acetyltransferases in distinct tumor types and new strategies for therapeutic disruption of subdomain and scaffolding activities of these multidomain proteins in cancer. Citation Format: Adam D. Durbin, Noha Shendy, Audrey Mercier, Yang Zhang, Melissa J. Bikowitz, Logan H. Sigua, Sarah Robinson, Tingjian Wang, Barbara Jonchere, A. Thomas Look, Mark W. Zimmerman, Martine Roussel, Brian J. Abraham, Ernst Schonbrunn, Kimberly Stegmaier, Jun Qi. Targeting EP300 and CBP for therapeutic benefit in pediatric solid tumors. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR005.

Acromegaly is a neuroendocrine disorder caused by excess secretion of GH by somatotroph tumor cells. It is often treated with somatostatin receptor (SSTR) 2 agonists, which suppress GH secretion. SOM230 is a somatostatin analogue that targets multiple SSTRs and was recently approved for patients with treatment-resistant acromegaly. Previous reports indicate that SOM230 may function as a biased agonist, suggesting that its ability to selectively activate SSTR-dependent signaling events may contribute to its therapeutic efficacy. To better understand how SOM230 modulates Sstr2A function, which is the most commonly expressed SSTR in somatotrophs, we used real-time assays to study SOM230-dependent signaling in rat pituitary tumor cells. We observed that SOM230 suppressed cAMP production in a Gαi-dependent manner, similar to conventional Sstr2A agonists. However, it did not cause receptor internalization as would be expected for an Sstr2A agonist. Surprisingly, SOM230 did not cause membrane hyperpolarization, which is an important mechanism by which Sstr2a activation suppresses intracellular calcium (Ca2+) accumulation and GH secretion. In fact, SOM230 inhibited the ability of conventional somatostatin analogues to control membrane potential. However, SOM230 still inhibited intracellular Ca2+ accumulation in a novel, Gβγ-dependent manner. These studies show that SOM230 exhibits strong agonist bias in regulating signaling pathways downstream of Sstr2A that control GH secretion., Using real-time measurements, we find that the SSTR agonist SOM230 exhibits bias toward signaling pathways linked to suppression of GH secretion in pituitary tumor cells.

Meiotic recombination ensures faithful segregation of homologous chromosomes during meiosis and generates genetic diversity in gametes. MEIOB (meiosis specific with OB domains), a meiosis-specific single-stranded DNA-binding homolog of replication protein A1 (RPA1), is essential for meiotic recombination. Here, we investigated the molecular mechanisms of MEIOB by characterizing its binding partners spermatogenesis associated 22 (SPATA22) and RPA. We find that MEIOB and SPATA22 form an obligate complex and contain defined interaction domains. The interaction between these two proteins is unusual in that nearly any deletion in the binding domains abolishes the interaction. Strikingly, a single residue D383 in MEIOB is indispensable for the interaction. The MEIOB/SPATA22 complex interacts with the RPA heterotrimeric complex in a collaborative manner. Furthermore, MEIOB and SPATA22 are recruited to induced DNA double-strand breaks (DSBs) together but not alone. These results demonstrate the cooperative property of the MEIOB-SPATA22 complex in its interaction with RPA and recruitment to DSBs.

Somatostatin (somatotropin release inhibiting factor) is an abundant neuropeptide, which acts on five subtypes of somatostatin receptor (SST1-SST5; nomenclature as agreed by the NC-IUPHAR Subcommittee on Somatostatin Receptors [89]). Activation of these receptors produces a wide range of physiological effects throughout the body including the inhibition of secretion of many hormones. Endogenous ligands for these receptors are somatostatin-14 (SRIF-14) and somatostatin-28 (SRIF-28). cortistatin-14 has also been suggested to be an endogenous ligand for somatostatin receptors [56].

Fluorescently labeled imaging agents can identify surgical margins in real-time to help achieve complete resections and minimize the likelihood of local recurrence. However, photon attenuation limits fluorescence-based imaging to superficial lesions or lesions that are a few millimeters beneath the tissue surface. Contrast agents that are dual-labeled with a radionuclide and fluorescent dye can overcome this limitation and combine quantitative, whole-body nuclear imaging with intraoperative fluorescence imaging. Using a multimodality chelation (MMC) scaffold, IRDye 800CW was conjugated to the clinically used somatostatin analog

Somatostatin analogs for the diagnosis and therapy of neuroendocrine tumors (NETs) have been used in clinical applications for more than two decades. Five somatostatin receptor subtypes have been identified and molecular mechanisms of somatostatin receptor signaling and regulation have been elucidated. These advances increased understanding of the biological role of each somatostatin receptor subtype, their distribution in NETs, as well as agonist-specific regulation of receptor signaling, internalization, and phosphorylation, particularly for the sst2 receptor subtype, which is the primary target of current somatostatin analog therapy for NETs. Various hypotheses exist to explain differences in patient responsiveness to somatostatin analog inhibition of tumor secretion and growth as well as differences in the development of tumor resistance to therapy. In addition, we now have a better understanding of the action of both first generation (octreotide, lanreotide, Octreoscan) and second generation (pasireotide) FDA-approved somatostatin analogs, including the biased agonistic character of some agonists. The increased understanding of somatostatin receptor pharmacology provides new opportunities to design more sophisticated assays to aid the future development of somatostatin analogs with increased efficacy.

Fluorescence-guided surgery is an emerging imaging technique that can enhance the ability of surgeons to detect tumors when compared with visual observation. To facilitate characterization, fluorescently labeled probes have been dual-labeled with a radionuclide to enable cross-validation with nuclear imaging. In this study, we selected the somatostatin receptor imaging agent DOTATOC as the foundation for developing a dual-labeled analog. We hypothesized that a customized dual-labeling approach with a multimodality chelation (MMC) scaffold would minimize steric effects of dye conjugation and retain agonist properties. Methods: An MMC conjugate (MMC-TOC) was synthesized on solid-phase and compared with an analog prepared using conventional methods (DA-TOC). Both analogs were conjugated to IRDye 800 using copper-free click chemistry. The resulting compounds, MMC(IR800)-TOC and DA(IR800)-TOC, were labeled with Cu and 64Cu and tested in vitro in somatostatin receptor subtype 2-overexpressing HEK-293 cells to assess agonist properties, and in AR42J rat pancreatic cancer cells to determine receptor binding characteristics. Multimodality imaging was performed in AR42J xenografts. Results: Cu-MMC(IR800)-TOC demonstrated higher potency for cyclic adenosine monophosphate inhibition (half maximal effective concentration [EC50]: 0.21 ± 0.18 vs. 1.38 ± 0.54 nM) and receptor internalization (EC50: 41.9 ± 29.8 vs. 455 ± 299 nM) than Cu-DA(IR800)-TOC. Radioactive uptake studies showed that blocking with octreotide caused a dose-dependent reduction in 64Cu-MMC(IR800)-TOC uptake whereas 64Cu-DA(IR800)-TOC was not affected. In vivo studies revealed higher tumor uptake for 64Cu-MMC(IR800)-TOC than 64Cu-DA(IR800)-TOC (5.2 ± 0.2 vs. 3.6 ± 0.4 percentage injected dose per gram). In vivo blocking studies with octreotide reduced tumor uptake of 64Cu-MMC(IR800)-TOC by 66%. Excretion of 64Cu-MMC(IR800)-TOC was primarily through the liver and spleen whereas 64Cu-DA(IR800)-TOC was cleared through the kidneys. Ex vivo analysis at 24 h confirmed PET/CT data by showing near-infrared fluorescence signal in tumors and a tumor-to-muscle ratio of 5.3 ± 0.8 as determined by γ-counting. Conclusion: The findings demonstrate that drug design affected receptor pharmacology and suggest that the MMC scaffold is a useful tool for the development of dual-labeled imaging agents.

High overexpression of somatostatin receptors in neuroendocrine tumors allows imaging and radiotherapy with radiolabeled somatostatin analogues. To ascertain whether a tumor is suitable for in vivo somatostatin receptor targeting, its somatostatin receptor expression has to be determined. There are specific indications for use of immunohistochemistry for the somatostatin receptor subtype 2A, but this has up to now been limited by the lack of an adequate reliable antibody. The aim of this study was to correlate immunohistochemistry using the new monoclonal anti-somatostatin receptor subtype 2A antibody UMB-1 with the gold standard in vitro method quantifying somatostatin receptor levels in tumor tissues. A UMB-1 immunohistochemistry protocol was developed, and tumoral UMB-1 staining levels were compared with somatostatin receptor binding site levels quantified with in vitro I-[Tyr]-octreotide autoradiography in 89 tumors. This allowed defining an immunohistochemical staining threshold permitting to distinguish tumors with somatostatin receptor levels high enough for clinical applications from those with low receptor expression. The presence of >10% positive tumor cells correctly predicted high receptor levels in 95% of cases. In contrast, absence of UMB-1 staining truly reflected low or undetectable somatostatin receptor expression in 96% of tumors. If 1% to 10% of tumor cells were stained, a weak staining intensity was suggestive of low somatostatin receptor levels. This study allows for the first time a reliable recommendation for eligibility of an individual patient for in vivo somatostatin receptor targeting based on somatostatin receptor immunohistochemistry. Under optimal methodological conditions, UMB-1 immunohistochemistry may be equivalent to in vitro receptor autoradiography.

The G(i)-coupled somatostatin 2A receptor (sst2A) mediates many of the neuromodulatory and neuroendocrine actions of somatostatin (SS) and is targeted by the SS analogs used to treat neuroendocrine tumors. As for other G protein-coupled receptors, agonists stimulate sst2A receptor phosphorylation on multiple residues, and phosphorylation at different sites has distinct effects on receptor internalization and uncoupling. To elucidate the spatial and temporal regulation of sst2A receptor phosphorylation, we examined agonist-stimulated phosphorylation of multiple receptor GPCR kinase sites using phospho-site-specific antibodies. SS increased receptor phosphorylation sequentially, first on Ser-341/343 and then on Thr-353/354, followed by receptor internalization. Reversal of receptor phosphorylation was determined by the duration of prior agonist exposure. In acutely stimulated cells, in which most receptors remained on the cell surface, dephosphorylation occurred only on Thr-353/354. In contrast, both Ser-341/343 and Thr-353/354 were rapidly dephosphorylated when cells were stimulated long enough to allow receptor internalization before agonist removal. Consistent with these observations, dephosphorylation of Thr-353/354 was not affected by either hypertonic sucrose or dynasore, which prevent receptor internalization, whereas dephosphorylation of Ser-341/343 was completely blocked. An okadaic acid- and fostriecin-sensitive phosphatase catalyzed the dephosphorylation of Thr-353/354 both intracellularly and at the cell surface. In contrast, dephosphorylation of Ser-341/343 was insensitive to these inhibitors. Our results show that the phosphorylation and dephosphorylation of neighboring GPCR kinase sites in the sst2A receptor are subject to differential spatial and temporal regulation. Thus, the pattern of receptor phosphorylation is determined by the duration of agonist stimulation and compartment-specific enzymatic activity.