Human iPSC-derived MSCs induce neurotrophic effects and improve metabolic activity in acute neuronal injury models.

Mesenchymal stromal cell (MSC) therapy has regenerative potentials to treat various pathological conditions including neurological diseases. MSCs isolated from various organs can differentiate into specific cell types to repair organ damages. However, their paracrine mechanisms are predicted to predominantly mediate their immunomodulatory, pro-angiogenic, and regenerative properties. While preclinical studies highlight the significant potential of MSC therapy in mitigating neurological damage from stroke and traumatic brain injury, the variability in clinical trial outcomes may stem from the inherent heterogeneity of somatic MSCs. Accumulating evidence has demonstrated that induced pluripotent stem cells (iPSCs) are an ideal alternative resource for the unlimited expansion and biomanufacturing of MSCs. Thus, we investigated how iPSC-derived MSCs (iMSCs) influence properties of iPSC-derived neurons. Our findings demonstrate that the secretome from iMSCs possesses neurotrophic effects, improving neuronal survival and promoting neuronal outgrowth and synaptic activity in vitro Additionally, the iMSCs enhance metabolic activity via mitochondrial respiration in neurons, both in vitro and in mouse models. Glycolytic pathways also increased following the administration of iMSC secretome to iPSC-derived neurons. Consistently, in vivo experiments showed that intravenous administration of iMSCs compensated for the elevated energetic demand in male mice with irradiation-induced brain injury by restoring synaptic metabolic activity during acute brain damage. 18F-FDG PET imaging also detected an increase in brain glucose uptake following iMSC administration. Together, our results highlight the potential of iMSC-based therapy in treating neuronal damage in various neurological disorders, while paving the way for future research and potential clinical applications of iMSCs in regenerative medicine.Significance Statement Regenerative biotherapeutics using MSCs have emerged as a promising intervention for treating various neurological diseases. Our study explored the potential beneficial effects of human iPSC-derived MSCs (iMSCs) on neurons. We demonstrated that molecules secreted into the culture medium by iMSCs enhance regenerative capabilities by improving neuronal survival, growth, and metabolic activity, as well as synaptic functions, in human iPSC-derived neurons. Mouse experiments also suggested the potential of iMSC therapy to mitigate synaptic mitochondrial dysfunction and enhance brain glucose uptake during acute radiation-induced brain injury, steps that contribute to restoring normal neuronal function. Our results highlight that iMSCs may be a promising alternative cell product for treating neuronal damage, overcoming the inconsistent efficacy of somatic MSCs due to cell variability.

Radiosynthesis and Preliminary Evaluation of [11C]SSI-4 for the Positron Emission Tomography Imaging of Stearoyl CoA Desaturase 1.

Stearoyl CoA desaturase 1 (SCD1) is the rate-limiting enzyme for converting saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs) and plays a key role in endogenous (de novo) fatty acid metabolism. Given that this pathway is broadly upregulated across many tumor types with an aggressive phenotype, SCD1 has emerged as a compelling target for cancer imaging and therapy. The ligand 2-(4-(2-chlorophenoxy)piperidine-1-carboxamido)-N-methylisonicotinamide (SSI-4) was identified as a potent and highly specific SCD1 inhibitor with a strong binding affinity for SCD1 at our laboratory. We herein report the radiosynthesis of [11C]SSI-4 and the preliminary biological evaluation including in vivo PET imaging of SCD1 in a human tumor xenograft model. Radiotracer [11C]SSI-4 was labeled at the carbamide position via the direct [11C]CO2 fixation on the Synthra MeIplus module in high molar activity and good radiochemical yield. In vitro cell uptake assays were performed with three hepatocellular carcinoma (HCC) cell lines and three renal cell carcinoma (RCC) cell lines. Additionally, in vivo small animal PET/CT imaging with [11C]SSI-4 and the biodistribution were carried out in a mouse model bearing HCC xenografts. Radiotracer [11C]SSI-4 afforded a 4.14 ± 0.44% (decay uncorrected, n = 10) radiochemical yield based on starting [11]CO2 radioactivity. The [11C]SSI-4 radiosynthesis time including HPLC purification and SPE formulation was 25 min from the end of bombardment to the end of synthesis (EOS). The radiochemical purity of [11C]SSI-4 was 98.45 ± 1.43% (n = 10) with a molar activity of 225.82 ± 33.54 GBq/µmol (6.10 ± 0.91 Ci/µmol) at the EOS. In vitro cell uptake study indicated all SSI-4 responsive HCC and RCC cell line uptakes demonstrate specific uptake and are blocked by standard compound SSI-4. Preliminary small animal PET/CT imaging study showed high specific uptake and block of [11C]SSI-4 uptake with co-injection of cold SSI-4 in high SCD1-expressing organs including lacrimal gland, brown fat, liver, and tumor. In summary, novel radiotracer [11C]SSI-4 was rapidly and automatedly radiosynthesized by direct [11C]CO2 fixation. Our preliminary biological evaluation results suggest [11C]SSI-4 could be a promising radiotracer for PET imaging of SCD1 overexpressing tumor tissues.

Ym155 Induces Oxidative Stress-Mediated DNA Damage and Cell Cycle Arrest, and Causes Programmed Cell Death in Anaplastic Thyroid Cancer Cells.

Anaplastic thyroid cancer (ATC) is one of the most lethal malignancies with a median survival time of about 4 months. Currently, there is no effective treatment, and the development of new therapies is an important and urgent issue for ATC patients. YM155 is a small molecule that was identified as the top candidate in a high-throughput screen of small molecule inhibitors performed against a panel of ATC cell lines by the National Cancer Institute. However, there were no follow-up studies investigating YM155 in ATC. Here, we determined the effects of YM155 on ATC and human primary benign thyroid cell (PBTC) survival with alamarBlue assay. Our data show that YM155 inhibited proliferation of ATC cell lines while sparing normal thyroid cells, suggesting a high therapeutic window. YM155-induced DNA damage was detected by measuring phosphorylation of γ-H2AX as a marker for DNA double-strand breaks. The formamidopyrimidine-DNA glycosylase (FPG)-modified alkaline comet assay in conjunction with reactive oxygen species (ROS) assay and glutathione (GSH)/glutathione (GSSG) assay suggests that YM155-mediated oxidative stress contributes to DNA damage. In addition, we provide evidence that YM155 causes cell cycle arrest in S phase and in the G2/M transition and causes apoptosis, as seen with flow cytometry. In this study, we show for the first time the multiple effects of YM155 in ATC cells, furthering a potential therapeutic approach for ATC.

Correlation of novel ALKATI with ALK immunohistochemistry and clinical outcomes in metastatic melanoma.

AIMS: Recently, a novel isoform of anaplastic lymphoma kinase, with alternative transcription initiation (ALKATI ), has been described in melanoma and is susceptible to targeted ALK-inhibitor therapy. Clinical outcomes of patients with ALKATI mutated melanoma as well as correlation with immunohistochemical (IHC) methods have not yet been described. METHODS AND RESULTS: Clinicopathological characteristics were abstracted for 324 patients with metastatic melanoma (MM). IHC, fluorescence in-situ hybridisation and RNA-based digital molecular analysis assays were performed on archival tissue from 173 stage III and 192 stage IV tumours. ALKATI was identified in 12.7 and 4.8% stage III and IV tumours, respectively. Discrete presentations of the ALKATI are seen: isolated ALKATI (n = 20) and mixed ALKATI (combined ALKATI and ALKWT ; n = 7). Isolated ALKWT expression (n = 4) was seen with no ALK fusions. Stage III patients showed improved survival with ALKATI expression compared to those with ALKWT or no expression [5-year survival 80, 95% confidence interval (CI) = 57-100% versus 43%, 95% CI = 34-55%, P = 0.013]. Clinicopathological characteristics were not statistically significant. Strong diffuse cytoplasmic staining of ALK IHC (n = 12) has a sensitivity of 52.2%, specificity 100%, PPV of 100% and NPV of 92.5% of detecting isolated ALKATI . CONCLUSION: Presence of ALKATI is a good prognostic indicator in MM. ALK IHC and digital molecular analysis can be incorporated into MM evaluation to identify patients with ALKATI for targeted therapy.

Cytokeratin-8 in Anaplastic Thyroid Carcinoma: More Than a Simple Structural Cytoskeletal Protein.

Anaplastic thyroid carcinoma (ATC) is almost universally fatal. Elevated keratin-8 (KRT8) protein expression is an established diagnostic cancer biomarker in several epithelial cancers (but not ATC). Several keratins, including KRT8, have been suggested to have a role in cell biology beyond that of structural cytoskeletal proteins. Here, we provide evidence that KRT8 plays a direct role in the growth of ATCs. Genomic and transcriptomic analysis of >5000 patients demonstrates that KRT8 mutation and copy number amplification are frequently evident in epithelial-derived cancers. Carcinomas arising from diverse tissues exhibit KRT8 mRNA and protein overexpression when compared to normal tissue levels. Similarly, in a panel of patient-derived ATC cell lines and patient tumors, KRT8 expression shows a similar pattern. sh-RNA-mediated KRT8 knockdown in these cell lines increases apoptosis, whereas forced overexpression of KRT8 confers resistance to apoptosis under peroxide-induced cell stress conditions. We further show that KRT8 protein binds to annexin A2, a protein known to mediate apoptosis as well as the redox pathway.

Establishing and characterizing patient-derived xenografts using pre-chemotherapy percutaneous biopsy and post-chemotherapy surgical samples from a prospective neoadjuvant breast cancer study.

BACKGROUND: Patient-derived xenografts (PDXs) are increasingly used in cancer research as a tool to inform cancer biology and drug response. Most available breast cancer PDXs have been generated in the metastatic setting. However, in the setting of operable breast cancer, PDX models both sensitive and resistant to chemotherapy are needed for drug development and prospective data are lacking regarding the clinical and molecular characteristics associated with PDX take rate in this setting. METHODS: The Breast Cancer Genome Guided Therapy Study (BEAUTY) is a prospective neoadjuvant chemotherapy (NAC) trial of stage I-III breast cancer patients treated with neoadjuvant weekly taxane+/-trastuzumab followed by anthracycline-based chemotherapy. Using percutaneous tumor biopsies (PTB), we established and characterized PDXs from both primary (untreated) and residual (treated) tumors. Tumor take rate was defined as percent of patients with the development of at least one stably transplantable (passed at least for four generations) xenograft that was pathologically confirmed as breast cancer. RESULTS: Baseline PTB samples from 113 women were implanted with an overall take rate of 27.4% (31/113). By clinical subtype, the take rate was 51.3% (20/39) in triple negative (TN) breast cancer, 26.5% (9/34) in HER2+, 5.0% (2/40) in luminal B and 0% (0/3) in luminal A. The take rate for those with pCR did not differ from those with residual disease in TN (p = 0.999) and HER2+ (p = 0.2401) tumors. The xenografts from 28 of these 31 patients were such that at least one of the xenografts generated had the same molecular subtype as the patient. Among the 35 patients with residual tumor after NAC adequate for implantation, the take rate was 17.1%. PDX response to paclitaxel mirrored the patients' clinical response in all eight PDX tested. CONCLUSIONS: The generation of PDX models both sensitive and resistant to standard NAC is feasible and these models exhibit similar biological and drug response characteristics as the patients' primary tumors. Taken together, these models may be useful for biomarker discovery and future drug development.

Stearoyl-CoA desaturase regulates sorafenib resistance via modulation of ER stress-induced differentiation.

BACKGROUND & AIMS: We investigated the functional role and clinical significance of stearoyl-CoA desaturase-1 (SCD1) mediated endoplasmic reticulum (ER) stress in regulating liver tumor-initiating cells (T-ICs) and sorafenib resistance, with the aim of developing a novel therapeutic strategy against hepatocellular carcinomas (HCCs). METHODS: We evaluated the clinic-pathological relevance of SCD1 and its correlation with sorafenib resistance in large cohorts of HCC clinical samples by qPCR and immunohistochemical analyses. Lentiviral-based overexpression and knockdown approaches were performed to characterize the functional roles of SCD1 in regulating liver T-ICs and sorafenib resistance. Molecular pathways mediating the phenotypic alterations were identified through RNA sequencing analysis and functional rescue experiments. The combinatorial effect of SCD1 inhibition and sorafenib was tested using a patient-derived tumor xenograft (PDTX) model. RESULTS: SCD1 overexpression was found in HCC, which was associated with shorter disease-free survival (p = 0.008, log rank test). SCD1 was found to regulate the populations of liver T-ICs; while its suppression by a SCD1 inhibitor suppressed liver T-ICs and sorafenib resistance. Interestingly, SCD1 was markedly upregulated in our established sorafenib-resistant PDTX model, and its overexpression predicts the clinical response of HCC patients to sorafenib treatment. Suppression of SCD1 forces liver T-ICs to differentiate via ER stress-induced unfolded protein response, resulting in an enhanced sensitivity to sorafenib. The PDTX#1 model, combined with sorafenib treatment and a novel SCD1 inhibitor (SSI-4), showed a maximal growth suppressive effect. CONCLUSIONS: SCD1-mediated ER stress regulates liver T-ICs and sorafenib sensitivity. Targeting SCD1 alone or in combination with sorafenib might be a novel personalized medicine against HCC. Lay summary: In this study, SCD1 was found to play a critical role in regulating liver tumor-initiating cells and sorafenib resistance through the regulation of ER stress-mediated differentiation. Targeting SCD1 in combination with sorafenib may be a novel therapeutic strategy against liver cancer.

Phase 2 trial of everolimus and letrozole in relapsed estrogen receptor-positive high-grade ovarian cancers.

OBJECTIVES: We report the results of a phase 2 clinical trial of the combination of everolimus and letrozole in patients with relapsed estrogen receptor-positive high-grade ovarian cancer. The trial's primary endpoint was the proportion of patients alive and progression-free after 12weeks of therapy with the combination of everolimus and letrozole. A 12-week PFS of 45% or greater was considered a positive result. The feasibility of generating patient-derived xenograft (PDX) models from biopsy specimens was also evaluated. METHODS: Eligibility criteria included relapsed estrogen receptor-positive ovarian, fallopian tube or primary peritoneal carcinomas with measurable disease, not previously treated with everolimus or AIs. Both platinum-resistant and sensitive tumors were included. Xenografts were created from image-guided tumor biopsies at baseline. Patients received oral everolimus 10mg daily and letrozole 2.5mg daily. RESULTS: Twenty patients were enrolled, 19 were evaluable. Nine out of 19 were alive, progression-free, and still on treatment at the 12week evaluation time-point (12-week PFS of 47%) with a median PFS of 3.9months (95% CI: 2.8-11.0). The median overall survival was 13.0months. Twelve patients (63%) experienced at least one grade 3 or worse adverse events. PDX tumor engraftment was feasible in the majority of patients (9 out of 17, 52.9%). CONCLUSIONS: The combination of everolimus and letrozole is associated with a promising 47% 12-week PFS rate in patients with ER-positive relapsed high-grade ovarian cancer with acceptable toxicity. PDX tumor models can be generated from biopsies of ovarian tumors.

Novel patient-derived xenograft mouse model for pancreatic acinar cell carcinoma demonstrates single agent activity of oxaliplatin.

BACKGROUND: Pancreatic acinar cell carcinoma (PACC) is a rare malignancy, accounting for <1 % of all pancreatic neoplasms. Very few retrospective studies are available to help guide management. We previously reported the case of a patient with metastatic PACC who achieved prolonged survival following doxorubicin treatment. Personalized treatment was based on molecular and in vitro data collected from primary cells developed from their liver metastasis. We now report the characterization of a patient derived tumor xenograft (PDTX) mouse model that originated from this patient's PACC liver metastasis. METHODS: Fragments of biopsy tissue (5 mm(3)) from PACC liver metastasis were implanted into athymic nude mice. Tumors were grown and passaged from the host mice into new mice to be tested for therapeutic response. Immuno-histochemical (IHC) biomarkers were used to confirm that the PDTX model represents human PACC. The antitumor activities of multiple drugs (5-FU, irinotecan, oxaliplatin, gemcitabine, bevacizumab, erlotinib, doxorubicin and imatinib) were tested. Tumor size was measured over 74 days or until they reached an endpoint volume of ~800 mm(3). Tests to measure serum lipase levels and histological analyses of tumor tissues were also conducted to assess PACC progression and re-differentiation. RESULTS: The model presented here expresses the same IHC markers found in human PACC. In the chemotherapy study, oxaliplatin produced a prolonged durable growth response associated with increased apoptosis, decreased serum lipase levels and increased healthy acinar cells. Bevacizumab also produced a significant growth response, but the effect was not prolonged as demonstrated by oxaliplatin treatment. The other chemotherapies had moderate to little effect, particularly after treatment ceased. Mutations in DNA repair genes are common in PACC and increase tumor susceptibility to oxaliplatin. To explore this we performed IHC and found no nuclear expression of BRCA2 in our model, indicating a mutation affecting nuclear localization. Gene sequencing confirms BRCA2 has a homozygous gene deletion on Exon 10, which frequently causes a protein truncation. CONCLUSIONS: In summary, we report the development and characterization of the first and only preclinical PACC PDTX model. Here we show sustained anti-tumor activity of single agent oxaliplatin, a compound that is more effective in tumors that harbor mutations in DNA repair genes. Our data shows that BRCA2 is mutated in our PACC model, which could contribute to the oxaliplatin sensitivity observed. Further studies on this rare PACC model can serve to elucidate other novel therapies, biomarkers, and molecular mechanisms of signaling and drug resistance.

High-grade endometrial stromal sarcoma as the initial presentation of an adult patient with Peutz-Jeghers Syndrome: a case report.

A 46-year-old female presents with a pelvic mass and is diagnosed as having a high-grade endometrial stromal sarcoma. During surgery, she is noted to have areas of intussusception of the small bowel secondary to large hamartomatous polyps. The patient had a previous history of small bowel obstruction secondary to what had been thought to be hyperplastic polyps but represented hamartomatous polyps on further review. Additional examination revealed the presence of subtle hyperpigmented macules on the fingers leading to a diagnosis of Peutz-Jeghers Syndrome (PJS). The diagnosis was confirmed by the presence of a germ-line STK11 mutation. Immunohistochemistry analysis of the tumor showed decreased expression of STK-11 as compared to one of the patient's hamartomatous polyps. Next generation sequencing of the tumor specimen failed to demonstrate a "second hit" somatic mutation in STK-11. This case represents the first case of endometrial stromal sarcoma associated with PJS and illustrates the importance of increased awareness of this condition among oncologists. PJS is associated with dysregulation of the mTOR pathway; treatment with an mTOR inhibitor was not effective in this case.

Foxo3a drives proliferation in anaplastic thyroid carcinoma through transcriptional regulation of cyclin A1: a paradigm shift that impacts current therapeutic strategies.

The Forkhead transcription factor, FoxO3a, is a known suppressor of primary tumor growth through transcriptional regulation of key genes regulating cell cycle arrest and apoptosis. In many types of cancer, in response to growth factor signaling, FoxO3a is phosphorylated by Akt, resulting in its exclusion from the nucleus. Here we show that FoxO3a remains nuclear in anaplastic thyroid carcinoma (ATC). This correlates with lack of Akt phosphorylation at serine473 in ATC cell lines and tissues of ATC patients, providing a potential explanation for nuclear FoxO3a. Mechanistically, nuclear FoxO3a promotes cell cycle progression by transcriptional upregulation of cyclin A1, promoting proliferation of human ATC cells. Silencing FoxO3a with a reverse genetics approach leads to downregulation of CCNA1 mRNA and protein. These combined data suggest an entirely novel function for FoxO3a in ATC promotion by enhancing cell cycle progression and tumor growth through transcriptional upregulation of cyclin A1. This is clinically relevant since we detected highly elevated CCNA1 mRNA and protein levels in tumor tissues of ATC patients. Our data indicate therapeutic inactivation of FoxO3a may lead to attenuation of tumor expansion in ATC. This new paradigm also suggests caution in relation to current dogma focused upon reactivation of FoxO3a as a therapeutic strategy against cancers harboring active PI3-K and Akt signaling pathways.

Zirconium- 89 Labeled Antibody K1-70 for PET Imaging of Thyroid-stimulating Hormone Receptor Expression in Thyroid Cancer.

PURPOSE: Thyroid-stimulating hormone receptor (TSHR) is a G-protein coupled receptor that is highly expressed on benign and malignant thyroid tissues. TSHR binding and activation has long been a component of thyroid cancer molecular imaging and radiotherapy, by promoting expression of the sodium-iodide symporter (NIS) and incorporation of I-131 into thyroid hormones. Here, we report the radiosynthesis and preclinical evaluation of a Zirconium-89 (89Zr) labeled TSHR antibody to serve as a positron emission tomography (PET) diagnostic correlate for therapeutic agents targeting TSHR without reliance on NIS. PROCEDURES: TSHR human monoclonal antibody K1-70 was conjugated to chelator desferrioxamine-p-benzyl-isothiocyanate, followed by labeling with Zr-89, yielding the radiotracer 89Zr-DFO-TSHR-Ab. The in vitro cellar uptake and binding affinity of 89Zr-DFO-TSHR-Ab were analyzed in three new TSHR stable overexpressing tumor cell lines and their corresponding wild types (WT) with low or no TSHR expression. 89Zr-DFO-TSHR-Ab PET/CT imaging of TSHR expression was evaluated in tumor mouse models bearing one TSHR-positive tumor and other negative control with or without the coinjection of antibody K1-70, and then verified by radiotracer biodistribution study and tumor immunohistochemistry (IHC). RESULTS: The conjugate DFO-TSHR-Ab was labeled with Zr-89 at 37 °C for 60 min and purified by PD-10 column in radiochemical yields of 68.8 ± 9.9%, radiochemical purities of 98.7 ± 0.8%, and specific activities of 19.1 ± 2.7 mCi/mg (n = 5). In vitro cell studies showed 89Zr-DFO-TSHR-Ab had significantly high uptake on TSHR expressing tumor cells with nanomolar affinity and high potency. Preclinical PET/CT imaging revealed that 89Zr-DFO-TSHR-Ab selectively detected TSHR expressing thyroid tumors and displayed improved in vivo performance with the coinjection of unlabeled TSHR antibody K1-70 leading to higher uptake in TSHR expressing tumors than parental WT tumors and physiologic tissues; this observation was confirmed by the biodistribution and immunostaining analyses. CONCLUSIONS: We synthesized 89Zr-labeled antibody K1-70 as a new radiopharmaceutical for PET imaging of TSHR. 89Zr-DFO-TSHR-Ab has high radioactive uptake and retention in TSHR expressing tumors and cleared quickly from most background tissues in mouse models. Our study demonstrated that 89Zr-DFO-TSHR-Ab has the potential for PET imaging of TSHR-positive thyroid cancer and monitoring TSHR-targeted therapy.

CRISPR screening identifies BET and mTOR inhibitor synergy in cholangiocarcinoma through serine glycine one carbon.

Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anticancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the 2 inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key enzymes PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single-agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.

Improving Circulation Half-Life of Therapeutic Candidate N-TIMP2 by Unfolded Peptide Extension.

Matrix metalloproteinases (MMPs) are significant drivers of many diseases, including cancer, and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous MMP inhibitors and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties for drug candidates, such as complete MMP inhibition, low toxicity, low immunogenicity, and high tissue permeability. However, a major challenge with TIMPs is their rapid clearance from the bloodstream due to their small size. This study explores a method for extending the plasma half-life of the N-terminal domain of TIMP2 (N-TIMP2) by appending it with a long, intrinsically unfolded tail containing Pro, Ala, and Thr (PATylation). We designed and produced two PATylated N-TIMP2 constructs with tail lengths of 100 and 200 amino acids (N-TIMP2-PAT100 and N-TIMP2-PAT200). Both constructs demonstrated higher apparent molecular weights and retained high inhibitory activity against MMP-9. N-TIMP2-PAT200 significantly increased plasma half-life in mice compared to the non-PATylated variant, enhancing its therapeutic potential. PATylation offers distinct advantages for half-life extension, such as fully genetic encoding, monodispersion, and biodegradability. It can be easily applied to N-TIMP2 variants engineered for high affinity and selectivity toward individual MMPs, creating promising candidates for drug development against MMP-related diseases.

Improving Circulation Half-Life of Therapeutic Candidate N-TIMP2 by Unfolded Peptide Extension.

Matrix Metalloproteinases (MMPs) are drivers of many diseases including cancer and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are human proteins that inhibit MMPs and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties of a drug candidate, such as complete MMP inhibition, low toxicity and immunogenicity, high tissue permeability and others. A major challenge with TIMPs, however, is their formulation and delivery, as these proteins are quickly cleared from the bloodstream due to their small size. In this study, we explore a new method for plasma half-life extension for the N-terminal domain of TIMP2 (N-TIMP2) through appending it with a long intrinsically unfolded tail containing a random combination of Pro, Ala, and Thr (PATylation). We design, produce and explore two PATylated N-TIMP2 constructs with a tail length of 100- and 200-amino acids (N-TIMP2-PAT100 and N-TIMP2-PAT200, respectively). We demonstrate that both PATylated N-TIMP2 constructs possess apparent higher molecular weights compared to the wild-type protein and retain high inhibitory activity against MMP-9. Furthermore, when injected into mice, N-TIMP2-PAT200 exhibited a significant increase in plasma half-life compared to the non-PATylated variant, enhancing the therapeutic potential of the protein. Thus, we establish that PATylation could be successfully applied to TIMP-based therapeutics and offers distinct advantages as an approach for half-life extension, such as fully genetic encoding of the gene construct, mono-dispersion, and biodegradability. Furthermore, PATylation could be easily applied to N-TIMP2 variants engineered to possess high affinity and selectivity toward individual MMP family members, thus creating attractive candidates for drug development against MMP-related diseases.

Thyroid-stimulating hormone receptor (TSHR) as a target for imaging differentiated thyroid cancer.

BACKGROUND: Of the half a million cases of thyroid cancer diagnosed annually, 95% are differentiated thyroid cancers. Although clinical guidelines recommend surgical resection followed by radioactive iodine ablation, loss of sodium-iodine symporter expression causes up to 20% of differentiated thyroid cancers to become radioactive iodine refractory. For patients with radioactive iodine refractory disease, there is an urgent need for new diagnostic and therapeutic approaches. We evaluated the thyroid-stimulating hormone receptor as a potential target for imaging of differentiated thyroid cancer. METHODS: We immunostained tissue microarrays containing 52 Hurthle cell carcinomas to confirm thyroid-stimulating hormone receptor expression. We radiolabeled chelator deferoxamine conjugated to recombinant human thyroid-stimulating hormone analog superagonist TR1402 with 89Zr (t1/2 = 78.4 h, ß+ =22.7%) to produce [89Zr]Zr-TR1402. We performed in vitro uptake assays in high-thyroid-stimulating hormone receptor and low-thyroid-stimulating hormone receptor-expressing THJ529T and FTC133 thyroid cancer cell lines. We performed in vivo positron emission tomography/computed tomography and biodistribution studies in male athymic nude mice bearing thyroid-stimulating hormone receptor-positive THJ529T tumors. RESULTS: Immunohistochemical analysis revealed 62% of patients (27 primary and 5 recurrent) were thyroid-stimulating hormone receptor membranous immunostain positive. In vitro uptake of 1nM [89Zr]Zr-TR1402 was 38 ± 17% bound/mg in thyroid-stimulating hormone receptor-positive THJ529T thyroid cancer cell lines compared to 3.2 ± 0.5 in the low-expressing cell line (P < .01), with a similar difference seen in FTC133 cell lines (P < .0001). In vivo and biodistribution studies showed uptake of [89Zr]Zr-TR1402 in thyroid-stimulating hormone receptor-expressing tumors, with a mean percentage of injected dose/g of 1.9 ± 0.4 at 3 days post-injection. CONCLUSION: Our observation of thyroid-stimulating hormone receptor expression in tissue microarrays and [89Zr]Zr-TR1402 accumulation in thyroid-stimulating hormone receptor-positive thyroid cancer cells and tumors suggests thyroid-stimulating hormone receptor is a promising target for imaging of differentiated thyroid cancer.

Patient-Derived Tumor Xenograft Study with CDK4/6 Inhibitor Plus AKT Inhibitor for the Management of Metastatic Castration-Resistant Prostate Cancer.

Metastatic castration-resistant prostate cancer (mCRPC) is an aggressive malignancy with poor outcomes. To investigate novel therapeutic strategies, we characterized three new metastatic prostate cancer patient derived-tumor xenograft (PDTX) models and developed 3D spheroids from each to investigate molecular targeted therapy combinations including CDK4/6 inhibitors (CDK4/6i) with AKT inhibitors (ATKi). Metastatic prostate cancer tissue was collected and three PDTX models were established and characterized using whole-exome sequencing. PDTX 3D spheroids were developed from these three PDTXs to show resistance patterns and test novel molecular-targeted therapies. CDK4/6i's were combined with AKTi's to assess synergistic antitumor response to prove our hypothesis that blockade of AKT overcomes drug resistance to CDK4/6i. This combination was evaluated in PDTX three-dimensional (3D) spheroids and in vivo experiments with responses measured by tumor volumes, PSA, and Ga-68 PSMA-11 PET-CT imaging. We demonstrated CDK4/6i's with AKTi's possess synergistic antitumor activity in three mCRPC PDTX models. These models have multiple unique pathogenic and deleterious genomic alterations with resistance to single-agent CDK4/6i's. Despite this, combination therapy with AKTi's was able to overcome resistance mechanisms. The IHC and Western blot analysis confirmed on target effects, whereas tumor volume, serum PSA ELISA, and radionuclide imaging demonstrated response to therapy with statistically significant SUV differences seen with Ga-68 PSMA-11 PET-CT. These preclinical data demonstrating antitumor synergy by overcoming single-agent CDK 4/6i as well as AKTi drug resistance provide the rational for a clinical trial combining a CDK4/6i with an AKTi in patients with mCRPC whose tumor expresses wild-type retinoblastoma 1.

The genomic and evolutionary landscapes of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations.