p53 Activates the Long Noncoding RNA Pvt1b to Inhibit Myc and Suppress Tumorigenesis.

The tumor suppressor p53 transcriptionally activates target genes to suppress cellular proliferation during stress. p53 has also been implicated in the repression of the proto-oncogene Myc, but the mechanism has remained unclear. Here, we identify Pvt1b, a p53-dependent isoform of the long noncoding RNA (lncRNA) Pvt1, expressed 50 kb downstream of Myc, which becomes induced by DNA damage or oncogenic signaling and accumulates near its site of transcription. We show that production of the Pvt1b RNA is necessary and sufficient to suppress Myc transcription in cis without altering the chromatin organization of the locus. Inhibition of Pvt1b increases Myc levels and transcriptional activity and promotes cellular proliferation. Furthermore, Pvt1b loss accelerates tumor growth, but not tumor progression, in an autochthonous mouse model of lung cancer. These findings demonstrate that Pvt1b acts at the intersection of the p53 and Myc transcriptional networks to reinforce the anti-proliferative activities of p53.

In Vivo Profiling with 18F-YJH08 Reveals Diverse Tissue Patterns of Antagonist/Glucocorticoid Receptor Interactions.

Demonstrating target engagement in vivo is an important milestone in drug development, both to establish on target, on tissue interactions but also to identify potentially undesirable off tissue binding. The glucocorticoid receptor (GR) is a long-studied yet vexing drug target that has recently re-emerged as a potential druggable driver of many solid tumor types including breast and prostate cancer, and several antagonists are currently in early phase clinical trials. Since GR is also ubiquitously expressed in normal tissues, understanding antagonist/GR interactions in normal tissues and tumor is crucial to defining a therapeutic index. Herein, we demonstrate that the GR radioligand 18F-YJH08 can map drug/GR engagement in vivo. Profiling target engagement in vivo showed that the GR antagonists RU486 (mifepristone) and CORT125281 engaged GR in fewer normal tissues compared to ORIC-101 or the agonist dexamethasone. Furthermore, 18F-YJH08 detected GR in human prostate cancer tumor models and measured receptor binding by RU486. In summary, these data show for the first time that antagonist/GR interactions can be measured in vivo with 18F-YJH08, a finding with clinical relevance as GR antagonists and 11C-YJH08 are currently in clinical trials.

Elevated labile iron in castration-resistant prostate cancer is targetable with ferrous iron-activatable antiandrogen therapy.

Clinical responses to second generation androgen signaling inhibitors (e.g., enzalutamide) in metastatic castration-resistant prostate cancer (mCRPC) are variable and transient, and are associated with dose limiting toxicities, including rare but severe CNS effects. We hypothesized that changes to iron metabolism coincident with more advanced disease might be leveraged for tumor-selective delivery of antiandrogen therapy. Using the recently described chemical probes SiRhoNox and 18F-TRX in mCRPC models, we found elevated Fe2+ to be a common feature of mCRPC in vitro and in vivo. We next synthesized ferrous-iron activatable drug conjugates of second and third-generation antiandrogens and found these conjugates possessed comparable or enhanced antiproliferative activity across mCRPC cell line models. Mouse pharmacokinetic studies showed that these prototype antiandrogen conjugates are stable in vivo and limited exposure to conjugate or free antiandrogen in the brain. Our results reveal elevated Fe2+ to be a feature of mCRPC that might be leveraged to improve the tolerability and efficacy of antiandrogen therapy.

Theranostic Targeting of CUB Domain-Containing Protein 1 (CDCP1) in Multiple Subtypes of Bladder Cancer.

PURPOSE: Despite recent approvals for checkpoint inhibitors and antibody-drug conjugates targeting NECTIN4 or TROP2, metastatic bladder cancer remains incurable and new treatment strategies are urgently needed. CUB domain-containing protein 1 (CDCP1) is a cell surface protein and promising drug target for many cancers. This study aimed to determine whether CDCP1 is expressed in bladder cancer and whether CDCP1 can be targeted for treatment with radiolabeled antibodies. EXPERIMENTAL DESIGN: CDCP1 expression was evaluated in four bladder cancer datasets (n = 1,047 biopsies). A tissue microarray of primary bladder cancer biopsies was probed for CDCP1 by IHC. CDCP1 expression was evaluated in patient-derived xenografts and cell lysates by immunoblot, flow cytometry, and saturation binding assays. Tumor detection in mouse bladder cancer models was tested using 89Zr-labeled 4A06, a monoclonal antibody targeting the ectodomain of CDCP1. 177Lu-4A06 was applied to mice bearing UMUC3 or HT-1376 xenografts to evaluate antitumor effects (CDCP1 expression in UMUC3 is 10-fold higher than HT-1376). RESULTS: CDCP1 was highest in the basal/squamous subtype, and CDCP1 was expressed in 53% of primary biopsies. CDCP1 was not correlated with pathologic or tumor stage, metastatic site, or NECTIN4 and TROP2 at the mRNA or protein level. CDCP1 ranged from 105 to 106 receptors per cell. Mechanism studies showed that RAS signaling induced CDCP1 expression. 89Zr-4A06 PET detected five human bladder cancer xenografts. 177Lu-4A06 inhibited the growth of UMUC3 and HT-1376 xenografts, models with high and moderate CDCP1 expression, respectively. CONCLUSIONS: These data establish that CDCP1 is expressed in bladder cancer, including TROP2 and NECTIN4-null disease, and suggest that bladder cancer can be treated with CDCP1-targeted radiotherapy.

CUB Domain-Containing Protein 1 (CDCP1) Is a Target for Radioligand Therapy in Castration-Resistant Prostate Cancer, including PSMA Null Disease.

PURPOSE: With the improvement in overall survival with 177Lu-PSMA 617, radioligand therapy (RLT) is now a viable option for patients with metastatic castration-resistant prostate cancer (mCRPC). However, responses are variable, in part due to low PSMA expression in 30% of patients. Herein, we evaluated whether the cell surface protein CUB domain-containing protein 1 (CDCP1) can be exploited to treat mCRPC with RLT, including in PSMA-low subsets. EXPERIMENTAL DESIGN: CDCP1 levels were evaluated using RNA sequencing from 119 mCRPC biopsies. CDCP1 levels were assessed in 17 post-enzalutamide- or abiraterone-treated mCRPC biopsies, 12 patient-derived xenografts (PDX), and prostate cancer cell lines. 4A06, a recombinant human antibody that targets the CDCP1 ectodomain, was labeled with Zr-89 or Lu-177 and tested in tumor-bearing mice. RESULTS: CDCP1 expression was observed in 90% of mCRPC biopsies, including small-cell neuroendocrine (SCNC) and adenocarcinomas with low FOLH1 (PSMA) levels. Fifteen of 17 evaluable mCRPC biopsies (85%) demonstrated membranous CDCP1 expression, and 4 of 17 (23%) had higher CDCP1 H-scores compared with PSMA. CDCP1 was expressed in 10 of 12 PDX samples. Bmax values of approximately 22,000, 6,200, and 2,800 fmol/mg were calculated for PC3, DU145, and C4-2B human prostate cancer cells, respectively. 89Zr-4A06 PET detected six human prostate cancer xenografts, including PSMA-low tumors. 177Lu-4A06 significantly suppressed growth of DU145 and C4-2B xenografts. CONCLUSIONS: The data provide the first evidence supporting CDCP1-directed RLT to treat mCRPC. Expanded studies are warranted to determine whether CDCP1 is a viable drug target for patients with mCPRC.

Ferronostics: Measuring Tumoral Ferrous Iron with PET to Predict Sensitivity to Iron-Targeted Cancer Therapies.

Although cancer has been known for decades to harbor an insatiable appetite for iron, only recently has the chemistry emerged to exploit this altered state therapeutically, by targeting the expanded cytosolic labile iron pool (LIP) of the cancer cell. The state of the art includes therapies that react with the LIP to produce cytotoxic radical species (in some cases also releasing drug payloads) and molecules that exacerbate LIP-induced oxidative stress to trigger ferroptosis. Effectively implementing LIP-targeted therapies in patients will require biomarkers to identify those tumors with the most elevated LIP and thus most likely to succumb to LIP-targeted interventions. Toward this goal, we tested whether tumor uptake of the novel LIP-sensing radiotracer 18F-TRX aligns with tumor sensitivity to LIP-targeted therapies. Methods:18F-TRX uptake was assessed in vivo among 10 subcutaneous and orthotopic human xenograft models. Glioma and renal cell carcinoma were prioritized because these tumors have the highest relative expression levels of STEAP3, the oxidoreductase that reduces ferric iron to the ferrous oxidation state, in the Broad Institute Cancer Cell Line Encyclopedia. The antitumor effects of the LIP-activated prodrug TRX-CBI, which releases the DNA alkylator CBI, were compared in mice bearing U251 or PC3 xenografts, tumors with high and intermediate levels of 18F-TRX uptake, respectively. Results:18F-TRX showed a wide range of tumor accumulation. An antitumor assessment study showed that the growth of U251 xenografts, the model with the highest 18F-TRX uptake, was potently inhibited by TRX-CBI. Moreover, the antitumor effects against U251 were significantly greater than those observed for PC3 tumors, consistent with the relative 18F-TRX-determined LIP levels in tumors before therapy. Lastly, a dosimetry study showed that the estimated effective human doses for adult male and female mice were comparable to those of other 18F-based imaging probes. Conclusion: We report the first evidence-to our knowledge-that tumor sensitivity to an LIP-targeted therapy can be predicted with a molecular imaging tool. More generally, these data bring a new dimension to the nuclear theranostic model by showing a requirement for imaging to quantify, in situ, the concentration of a metastable bioanalyte toward predicting tumor drug sensitivity.

Temporal Control of the TGF-ß Signaling Network by Mouse ESC MicroRNA Targets of Different Affinities.

Although microRNAs (miRNAs) function in the control of embryonic stem cell (ESC) pluripotency, a systems-level understanding is still being developed. Through the analysis of progressive Argonaute (Ago)-miRNA depletion and rescue, including stable Ago knockout mouse ESCs, we uncover transforming growth factor beta (TGF-ß) pathway activation as a direct and early response to ESC miRNA reduction. Mechanistically, we link the derepression of weaker miRNA targets, including TGF-ß receptor 1 (Tgfbr1), to the sensitive TGF-ß pathway activation. In contrast, stronger miRNA targets impart a more robust repression, which dampens concurrent transcriptional activation. We verify such dampened induction for TGF-ß antagonist Lefty. We find that TGF-ß pathway activation contributes to the G1 cell-cycle accumulation of miRNA-deficient ESCs. We propose that miRNA target affinity is a determinant of the temporal response to miRNA changes, which enables the coordination of gene network responses.