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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. 18 F-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., (Copyright © 2024 Kawatani et al.)

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 ( 89 Zr) 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 89 Zr-DFO-TSHR-Ab. The in vitro cellar uptake and binding affinity of 89 Zr-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. 89 Zr-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 89 Zr-DFO-TSHR-Ab had significantly high uptake on TSHR expressing tumor cells with nanomolar affinity and high potency. Preclinical PET/CT imaging revealed that 89 Zr-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 89 Zr-labeled antibody K1-70 as a new radiopharmaceutical for PET imaging of TSHR. 89 Zr-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 89 Zr-DFO-TSHR-Ab has the potential for PET imaging of TSHR-positive thyroid cancer and monitoring TSHR-targeted therapy., (© 2024. The Author(s), under exclusive licence to World Molecular Imaging Society.)

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-PAT 100 and N-TIMP2-PAT 200 ). Both constructs demonstrated higher apparent molecular weights and retained high inhibitory activity against MMP-9. N-TIMP2-PAT 200 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., Competing Interests: The authors declare no conflicts of interest.

Thyroid cancer is the most common endocrine cancer, with differentiated thyroid cancers (DTCs) accounting for 95% of diagnoses. While most DTC patients are diagnosed and treated with radioiodine (RAI), up to 20% of DTC patients become RAI refractory (RAI-R). RAI-R patients have significantly reduced survival rates compared to patients who remain RAI-avid. This study explores [ 89 Zr]Zr-TR1402 as a thyroid-stimulating hormone receptor (TSHR)-targeted PET radiopharmaceutical for DTC. [ 89 Zr]Zr-TR1402 was synthesized with a molar activity of 25.9 MBq/nmol by conjugating recombinant human TSH (rhTSH) analogue TR1402 to chelator p-SCN-Bn-deferoxamine (DFO) in a molar ratio of 3:1 (DFO/TR1402) and radiolabeling with 89 Zr ( t 1/2 = 78.4 h, β + = 22.7%). As TSHR is absent in commonly available DTC-derived cell lines, TSHR was reintroduced via stable transduction by delivering a lentivirus containing the full-length coding region of the human TSHR gene. Receptor-mediated uptake of [ 89 Zr]Zr-TR1402 was evaluated in vitro in stably transduced TSHR+ and wild-type TSHR- DTC cell lines. In vivo PET imaging was performed on Days 1-3 postinjection in male and female athymic nude mice bearing TSHR+ and TSHR- xenografts, along with ex vivo biodistribution on Day 3 postinjection. In vitro uptake of 1 nM [ 89 Zr]Zr-TR1402 was significantly higher in TSHR+ THJ529T ( P < 0.0001) and FTC133 ( P < 0.01) cells than in TSHR- THJ529T and FTC133 cells. This uptake was shown to be specific in both TSHR+ THJ529T ( P < 0.0001) and TSHR+ FTC133 ( P < 0.0001) cells by blocking uptake with 250 nm DFO-TR1402. In vivo PET imaging showed accumulation of [ 89 Zr]Zr-TR1402 in TSHR+ tumors, which was the highest on Day 1. In the male FTC133 xenograft model, ex vivo biodistribution confirmed a significant difference ( P < 0.001) in uptake between FTC133+ (1.3 ± 0.1%ID/g) and FTC133- (0.8 ± 0.1%ID/g) tumors. A significant difference ( P < 0.05) in uptake was also seen in the male THJ529T xenograft model between THJ529T+ (1.8 ± 0.6%ID/g) and THJ529T- (0.8 ± 0.4%ID/g) tumors. The in vitro and in vivo accumulation of [ 89 Zr]Zr-TR1402 in TSHR-expressing DTC cell lines support the continued preclinical optimization of this approach.

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., (©2024 American Association for Cancer Research.)

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., Competing Interests: Declaration of interests All authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

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 89 Zr (t 1/2  = 78.4 h, β +  =22.7%) to produce [ 89 Zr]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 [ 89 Zr]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 [ 89 Zr]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 [ 89 Zr]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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: CDK4/6 inhibitors (CDK4/6i) have been established as standard treatment against advanced Estrogen Receptor-positive breast cancers. These drugs are being tested against several cancers, including in combinations with other therapies. We identified the T172-phosphorylation of CDK4 as the step determining its activity, retinoblastoma protein (RB) inactivation, cell cycle commitment and sensitivity to CDK4/6i. Poorly differentiated (PDTC) and anaplastic (ATC) thyroid carcinomas, the latter considered one of the most lethal human malignancies, represent major clinical challenges. Several molecular evidence suggest that CDK4/6i could be considered for treating these advanced thyroid cancers., Methods: We analyzed by two-dimensional gel electrophoresis the CDK4 modification profile and the presence of T172-phosphorylated CDK4 in a collection of 98 fresh-frozen tissues and in 21 cell lines. A sub-cohort of samples was characterized by RNA sequencing and immunohistochemistry. Sensitivity to CDK4/6i (palbociclib and abemaciclib) was assessed by BrdU incorporation/viability assays. Treatment of cell lines with CDK4/6i and combination with BRAF/MEK inhibitors (dabrafenib/trametinib) was comprehensively evaluated by western blot, characterization of immunoprecipitated CDK4 and CDK2 complexes and clonogenic assays., Results: CDK4 phosphorylation was detected in all well-differentiated thyroid carcinomas (n=29), 19/20 PDTC, 16/23 ATC and 18/21 thyroid cancer cell lines, including 11 ATC-derived ones. Tumors and cell lines without phosphorylated CDK4 presented very high p16 CDKN2A levels, which were associated with proliferative activity. Absence of CDK4 phosphorylation in cell lines was associated with CDK4/6i insensitivity. RB1 defects (the primary cause of intrinsic CDK4/6i resistance) were not found in 5/7 tumors without detectable phosphorylated CDK4. A previously developed 11-gene expression signature identified the likely unresponsive tumors, lacking CDK4 phosphorylation. In cell lines, palbociclib synergized with dabrafenib/trametinib by completely and permanently arresting proliferation. These combinations prevented resistance mechanisms induced by palbociclib, most notably Cyclin E1-CDK2 activation and a paradoxical stabilization of phosphorylated CDK4 complexes., Conclusion: Our study supports further clinical evaluation of CDK4/6i and their combination with anti-BRAF/MEK therapies as a novel effective treatment against advanced thyroid tumors. Moreover, the complementary use of our 11 genes predictor with p16/KI67 evaluation could represent a prompt tool for recognizing the intrinsically CDK4/6i insensitive patients, who are potentially better candidates to immediate chemotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pita, Raspé, Coulonval, Decaussin-Petrucci, Tarabichi, Dom, Libert, Craciun, Andry, Wicquart, Leteurtre, Trésallet, Marlow, Copland, Durante, Maenhaut, Cavaco, Dumont, Costante and Roger.)

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 [ 11 C]SSI-4 and the preliminary biological evaluation including in vivo PET imaging of SCD1 in a human tumor xenograft model. Radiotracer [ 11 C]SSI-4 was labeled at the carbamide position via the direct [ 11 C]CO 2 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 [ 11 C]SSI-4 and the biodistribution were carried out in a mouse model bearing HCC xenografts. Radiotracer [ 11 C]SSI-4 afforded a 4.14 ± 0.44% (decay uncorrected, n = 10) radiochemical yield based on starting [ 11 ]CO 2 radioactivity. The [ 11 C]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 [ 11 C]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 [ 11 C]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 [ 11 C]SSI-4 was rapidly and automatedly radiosynthesized by direct [ 11 C]CO 2 fixation. Our preliminary biological evaluation results suggest [ 11 C]SSI-4 could be a promising radiotracer for PET imaging of SCD1 overexpressing tumor tissues.

Bromodomains (BD) are epigenetic readers of histone acetylation involved in chromatin remodeling and transcriptional regulation of several genes including protooncogene cellular myelocytomatosis (c-Myc). c-Myc is difficult to target directly by agents due to its disordered alpha helical protein structure and predominant nuclear localization. The epigenetic targeting of c-Myc by BD inhibitors is an attractive therapeutic strategy for prostate cancer (PC) associated with increased c-Myc upregulation with advancing disease. MT-1 is a bivalent BD inhibitor that is 100-fold more potent than the first-in-class BD inhibitor JQ1. MT-1 decreased cell viability and causes cell cycle arrest in G0/G1 phase in castration-sensitive and resistant PC cell lines in a dose-dependent fashion. The inhibition of c-Myc function by MT-1 was molecularly corroborated by the de-repression of Protein Kinase D1 (PrKD) and increased phosphorylation of PrKD substrate proteins: threonine 120, serine 11, and serine 216 amino acid residues in β-Catenin, snail, and cell division cycle 25c (CDC25c) proteins, respectively. The treatment of 3D cell cultures derived from three unique clinically annotated heavily pretreated patient-derived PC xenografts (PDX) mice models with increasing doses of MT-1 demonstrated the lowest IC 50 in tumors with c-Myc amplification and clinically resistant to Docetaxel, Cabazitaxel, Abiraterone, and Enzalutamide. An intraperitoneal injection of either MT-1 or in combination with 3jc48-3, an inhibitor of obligate heterodimerization with MYC-associated protein X (MAX), in mice implanted with orthotopic PC PDX, decreased tumor growth. This is the first pre-clinical study demonstrating potential utility of MT-1 in the treatment of PC with c-Myc dysregulation.

Targetable drivers governing 5-fluorouracil and cisplatin (5FU + CDDP) resistance remain elusive due to the paucity of physiologically and therapeutically relevant models. Here, we establish 5FU + CDDP resistant intestinal subtype GC patient-derived organoid lines. JAK/STAT signaling and its downstream, adenosine deaminases acting on RNA 1 (ADAR1), are shown to be concomitantly upregulated in the resistant lines. ADAR1 confers chemoresistance and self-renewal in an RNA editing-dependent manner. WES coupled with RNA-seq identify enrichment of hyper-edited lipid metabolism genes in the resistant lines. Mechanistically, ADAR1-mediated A-to-I editing on 3'UTR of stearoyl-CoA desaturase (SCD1) increases binding of KH domain-containing, RNA-binding, signal transduction-associated 1 (KHDRBS1), thereby augmenting SCD1 mRNA stability. Consequently, SCD1 facilitates lipid droplet formation to alleviate chemotherapy-induced ER stress and enhances self-renewal through increasing β-catenin expression. Pharmacological inhibition of SCD1 abrogates chemoresistance and tumor-initiating cell frequency. Clinically, high proteomic level of ADAR1 and SCD1, or high SCD1 editing/ADAR1 mRNA signature score predicts a worse prognosis. Together, we unveil a potential target to circumvent chemoresistance., (© 2023. The Author(s).)

Purpose: Development of B-cell lymphoma 2 (BCL-2)-specific inhibitors poses unique challenges in drug design because of BCL-2 homology domain 3 (BH3) shared homology between BCL-2 family members and the shallow surface of their protein-protein interactions. We report herein discovery and extensive preclinical investigation of lisaftoclax (APG-2575)., Experimental Design: Computational modeling was used to design "lead" compounds. Biochemical binding, mitochondrial BH3 profiling, and cell-based viability or apoptosis assays were used to determine the selectivity and potency of BCL-2 inhibitor lisaftoclax. The antitumor effects of lisaftoclax were also evaluated in several xenograft models., Results: Lisaftoclax selectively binds BCL-2 (Ki < 0.1 nmol/L), disrupts BCL-2:BIM complexes, and compromises mitochondrial outer membrane potential, culminating in BAX/BAK-dependent, caspase-mediated apoptosis. Lisaftoclax exerted strong antitumor activity in hematologic cancer cell lines and tumor cells from patients with chronic lymphocytic leukemia, multiple myeloma, or Waldenström macroglobulinemia. After lisaftoclax treatment, prodeath proteins BCL-2‒like protein 11 (BIM) and Noxa increased, and BIM translocated from cytosol to mitochondria. Consistent with these apoptotic activities, lisaftoclax entered malignant cells rapidly, reached plateau in 2 hours, and significantly downregulated mitochondrial respiratory function and ATP production. Furthermore, lisaftoclax inhibited tumor growth in xenograft models, correlating with caspase activation, poly (ADP-ribose) polymerase 1 cleavage, and pharmacokinetics of the compound. Lisaftoclax combined with rituximab or bendamustine/rituximab enhanced antitumor activity in vivo., Conclusions: These findings demonstrate that lisaftoclax is a novel, orally bioavailable BH3 mimetic BCL-2-selective inhibitor with considerable potential for the treatment of certain hematologic malignancies., (©2022 American Association for Cancer Research.)

Branched-chain amino acid transaminase 1 (BCAT1) is upregulated selectively in human isocitrate dehydrogenase (IDH) wildtype (WT) but not mutant glioblastoma multiforme (GBM) and promotes IDHWT GBM growth. Through a metabolic synthetic lethal screen, we report here that α-ketoglutarate (AKG) kills IDHWT GBM cells when BCAT1 protein is lost, which is reversed by reexpression of BCAT1 or supplementation with branched-chain α-ketoacids (BCKA), downstream metabolic products of BCAT1. In patient-derived IDHWT GBM tumors in vitro and in vivo, cotreatment of BCAT1 inhibitor gabapentin and AKG resulted in synthetic lethality. However, AKG failed to evoke a synthetic lethal effect with loss of BCAT2, BCKDHA, or GPT2 in IDHWT GBM cells. Mechanistically, loss of BCAT1 increased the NAD+/NADH ratio but impaired oxidative phosphorylation, mTORC1 activity, and nucleotide biosynthesis. These metabolic alterations were synergistically augmented by AKG treatment, thereby causing mitochondrial dysfunction and depletion of cellular building blocks, including ATP, nucleotides, and proteins. Partial restoration of ATP, nucleotides, proteins, and mTORC1 activity by BCKA supplementation prevented IDHWT GBM cell death conferred by the combination of BCAT1 loss and AKG. These findings define a targetable metabolic vulnerability in the most common subset of GBM that is currently incurable., Significance: Metabolic synthetic lethal screening in IDHWT glioblastoma defines a vulnerability to ΑΚG following BCAT1 loss, uncovering a therapeutic strategy to improve glioblastoma treatment. See related commentary by Meurs and Nagrath, p. 2354., (©2022 American Association for Cancer Research.)

Anaplastic thyroid cancer (ATC) is among the most aggressive of human cancers, and currently there are few effective treatments for most patients. YM155, first identified as a survivin inhibitor, was highlighted in a high-throughput screen performed by the National Cancer Institute, killing ATC cells in vitro and in vivo. However, there was no association between survivin expression and response to YM155 in clinical trials, and YM155 has been mostly abandoned for development despite favorable pharmacokinetic and toxicity profiles. Currently, alternative mechanisms are being explored for YM155 by a number of groups. In this study, ATC patient samples show overexpression of topoisomerase Top2α compared with benign thyroid samples and to differentiated thyroid cancers. ATC cell lines that overexpress Top2α are more sensitive to YM155. We created a YM155-resistant cell line, which shows decreased expression of Top2α and is resensitized with Top2α overexpression. Molecular modeling predicts binding for YM155 in the Top2α ATP-binding site and identifies key amino acids for YM155-Top2α interaction. A Top2α mutant abrogates the effect of YM155, confirming the contribution of Top2α to YM155 mechanism of action. Our results suggest a novel mechanism of action for YM155 and may represent a new therapeutic approach for the treatment of ATC., (©2022 American Association for Cancer Research.)

Conventional imaging has been the standard imaging modality for assessing prostate cancer recurrence and is utilized to determine treatment response to therapy. Molecular imaging with PSMA PET-CT has proven to be more accurate, sensitive, and specific at identifying pelvic or distant metastatic disease, resulting in earlier diagnosis of advanced disease. Since advanced disease may not be seen on conventional imaging, due to its lower sensitivity, but can be identified by molecular imaging, this reveals that metastatic prostate cancer occurs on a continuum from negative PSMA PET-CT and negative conventional imaging to positive PSMA PET-CT and positive conventional imaging. Understanding this continuum, the accuracy of these modalities, and treatment related outcomes based on imaging, will allow the clinician to counsel patients on management. This review will highlight the differences in conventional and molecular imaging in prostate cancer and how PSMA PET-CT can be used for the management of prostate cancer patients in different clinical scenarios, while providing cautionary notes for overtreatment.

Purpose: Patient-derived xenografts (PDX) are a research tool for studying cancer biology and drug response phenotypes. While engraftment rates are higher for tumors with more aggressive characteristics, it is uncertain whether engraftment is prognostic for cancer recurrence., Patients and Methods: In a prospective study of patients with breast cancer treated with neoadjuvant chemotherapy (NAC) with taxane ± trastuzumab followed by anthracycline-based chemotherapy, we report the association between breast cancer events and PDX engraftment using tumors derived from treatment naïve (pre-NAC biopsies from 113 patients) and treatment resistant (post-NAC at surgery from 34 patients). Gray test was used to assess whether the cumulative incidence of a breast cancer event differs with respect to either pre-NAC PDX engraftment or post-NAC PDX engraftment., Results: With a median follow-up of 5.7 years, the cumulative incidence of breast cancer relapse did not differ significantly according to pre-NAC PDX engraftment (5-year rate: 13.6% vs. 13.4%; P = 0.89). However, the incidence of a breast event was greater for patients with post-NAC PDX engraftment (5-year rate: 50.0% vs. 19.6%), but this did not achieve significance ( P = 0.11)., Conclusions: In treatment-naïve breast cancer receiving standard NAC, PDX engraftment was not prognostic for breast cancer recurrence. Further study is needed to establish whether PDX engraftment in the treatment-resistant setting is prognostic for cancer recurrence., (©2021 American Association for Cancer Research.)

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.

Background: Bone metastatic prostate cancer (BMPCa), despite the initial responsiveness to androgen deprivation therapy (ADT), inevitably becomes resistant. Recent clinical trials with upfront treatment of ADT combined with chemotherapy or novel hormonal therapies (NHTs) have extended overall patient survival. These results indicate that there is significant potential for the optimization of standard-of-care therapies to delay the emergence of progressive metastatic disease., Methods: Here, we used data extracted from human bone metastatic biopsies pre- and post-abiraterone acetate/prednisone to generate a mathematical model of bone metastatic prostate cancer that can unravel the treatment impact on disease progression. Intra-tumor heterogeneity in regard to ADT and chemotherapy resistance was derived from biopsy data at a cellular level, permitting the model to track the dynamics of resistant phenotypes in response to treatment from biological first-principles without relying on data fitting. These cellular data were mathematically correlated with a clinical proxy for tumor burden, utilizing prostate-specific antigen (PSA) production as an example., Results: Using this correlation, our model recapitulated the individual patient response to applied treatments in a separate and independent cohort of patients (n = 24), and was able to estimate the initial resistance to the ADT of each patient. Combined with an intervention-decision algorithm informed by patient-specific prediction of initial resistance, we propose to optimize the sequence of treatments for each patient with the goal of delaying the evolution of resistant disease and limit cancer cell growth, offering evidence for an improvement against retrospective data., Conclusions: Our results show how minimal but widely available patient information can be used to model and track the progression of BMPCa in real time, offering a clinically relevant insight into the patient-specific evolutionary dynamics of the disease and suggesting new therapeutic options for intervention., Trial Registration: NCT # 01953640., Funding: Funded by an NCI U01 (NCI) U01CA202958-01 and a Moffitt Team Science Award. CCL and DB were partly funded by an NCI PSON U01 (U01CA244101). AA was partly funded by a Department of Defense Prostate Cancer Research Program (W81XWH-15-1-0184) fellowship. LC was partly funded by a postdoctoral fellowship (PF-13-175-01-CSM) from the American Cancer Society.

Immune cells can metabolize glucose, amino acids, and fatty acids (FAs) to generate energy. The roles of different FA species and their impacts on humoral immunity remain poorly understood. Here, we report that proliferating B cells require monounsaturated FAs (MUFAs) to maintain mitochondrial metabolism and mTOR activity and to prevent excessive autophagy and endoplasmic reticulum (ER) stress. Furthermore, B cell-extrinsic stearoyl-CoA desaturase (SCD) activity generates MUFA to support early B cell development and germinal center (GC) formation in vivo during immunization and influenza infection. Thus, SCD-mediated MUFA production is critical for humoral immunity., Competing Interests: Declaration of Interests H.Z. and J.A.C. have a pending patent application regarding use of the SCD inhibitor SSI-4., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Chloroethylagelastatin A (CEAA) is an analogue of agelastatin A (AA), a natural alkaloid derived from a marine sponge. It is under development for therapeutic use against brain tumors as it has excellent central nervous system (CNS) penetration and pre-clinical therapeutic activity against brain tumors. Recently, AA was shown to inhibit protein synthesis by binding to the ribosomal A-site. In this study, we developed a novel virtual screening platform to perform a comprehensive screening of various AA analogues showing that AA analogues with proven therapeutic activity including CEAA have significant ribosomal binding capacity whereas therapeutically inactive analogues show poor ribosomal binding and revealing structural fingerprint features essential for drug-ribosome interactions. In particular, CEAA was found to have greater ribosomal binding capacity than AA. Biological tests showed that CEAA binds the ribosome and contributes to protein synthesis inhibition. Our findings suggest that CEAA may possess ribosomal inhibitor activity and that our virtual screening platform may be a useful tool in discovery and development of novel ribosomal inhibitors.

Aims: Recently, a novel isoform of anaplastic lymphoma kinase, with alternative transcription initiation (ALK ATI ), has been described in melanoma and is susceptible to targeted ALK-inhibitor therapy. Clinical outcomes of patients with ALK ATI 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. ALK ATI was identified in 12.7 and 4.8% stage III and IV tumours, respectively. Discrete presentations of the ALK ATI are seen: isolated ALK ATI (n = 20) and mixed ALK ATI (combined ALK ATI and ALK WT ; n = 7). Isolated ALK WT expression (n = 4) was seen with no ALK fusions. Stage III patients showed improved survival with ALK ATI expression compared to those with ALK WT 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 ALK ATI ., Conclusion: Presence of ALK ATI is a good prognostic indicator in MM. ALK IHC and digital molecular analysis can be incorporated into MM evaluation to identify patients with ALK ATI for targeted therapy., (© 2020 John Wiley & Sons Ltd.)

Objective: We describe a phase II clinical trial of the combination of ribociclib and letrozole for treatment of relapsed oestrogen receptor (ER)-positive ovarian cancer (OC) and endometrial cancer (EC). The primary endpoint was the proportion of patients alive, progression-free survival (PFS), and still on treatment at 12 weeks (PFS12), with 45% or greater considered positive., Methods: Patients with measurable, relapsed ER-positive OC or EC (platinum-sensitive or resistant) were eligible and treated with 400 mg of oral ribociclib and 2.5 mg of oral letrozole daily. Patient-derived xenografts (PDXs) were created from imaging-guided tumour biopsies., Results: Forty patients (20 OC and 20 EC) were enrolled. A PFS12 of 55% was observed in the EC cohort and 50% in the OC cohort. A PFS greater or equal to 24 weeks (PFS24) was seen in 20% (4/20) of the OC cohort and 35% (7/20) of the EC cohort. The greatest benefit was seen in low-grade serous OC (LGSOC) (3/3, 100% PFS24) and grades 1 and 2 EC (5/11, 45% PFS24). All three LGSOC patients obtained at least a partial response lasting for over 2 years, with two of the three patients still on treatment. PDX tumour engraftment was feasible in 45% of patients. Positive survival effects of the combination of ribociclib and letrozole were observed in two of three EC PDX models., Conclusion: Ribociclib and letrozole have promising clinical activity in relapsed ER-positive OC and EC, particularly in LGSOC and relapsed ER-positive grade 1 and 2 EC. Generation of PDX models is feasible with positive survival effects observed in EC models., Trial Registration Number: ClinicalTrials.gov registry (NCT02657928)., Competing Interests: Competing interests: GC-O and SJW report grants from Novartis during the conduct of the study. MSB reports grants from Immune Design, Pharmacyclics, Marker Therapeutics, Merck, Genentech and Bristol Myers Squibb, outside the submitted work. We certify that the other authors have no financial affiliation/interest (eg, employment, stock holdings, consultant arrangements, honoraria in the subject matter, materials or products) mentioned in this manuscript., (© Author (s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. Published by BMJ on behalf of the European Society for Medical Oncology.)

Prostate cancer (PCa) is a common cancer in men that is curable prior to metastasis, when its prognosis worsens. Chondroitin sulfate (CS) is found in the extracellular matrix of normal prostate tissue and PCa, with greater content in metastatic PCa. Biomaterial scaffolds containing CS have yet to be evaluated for tumor microenvironment applications. Three-dimensional porous chitosan-CS (C-CS) scaffolds were developed and evaluated for PCa culture. Three C-CS scaffold compositions were prepared with 4 w/v% chitosan and 0.1, 0.5, and 1.0 w/v% CS and named 4-0.1, 4-0.5, and 4-1, respectively. The C-CS scaffolds had 90-95% porosity, average pore sizes between 143 and 166 μm, and no significant difference in scaffold stiffness. PC-3 and 22Rv1 PCa cells were cultured on the C-CS scaffolds to study the effect of CS on PCa growth and epithelial to mesenchymal transition (EMT). All C-CS scaffold compositions supported PCa growth and the 4-1 scaffolds had the greatest cell numbers for both PC-3 and 22Rv1. The C-CS scaffolds promoted upregulated EMT marker expression compared to 2D cultures with the greatest EMT marker expression in 4-1 scaffolds. Increasing CS concentration promoted upregulated vimentin expression in PC-3 cultures and N-cadherin and MMP-2 expression in 22Rv1 cultures. C-CS scaffolds promoted docetaxel drug resistance in PC-3 and 22Rv1 cultures and the 4-1 scaffold cultures had the greatest drug resistance. These results indicate that C-CS scaffolds are a promising in vitro platform for PCa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Clear-cell renal cell carcinoma (ccRCC) presents challenges to clinical management because of late-stage detection, treatment resistance, and frequent disease recurrence. Metabolically, ccRCC has a well-described Warburg effect utilization of glucose, but how this affects complex carbohydrate synthesis and alterations to protein and cell surface glycosylation is poorly defined. Using an imaging mass spectrometry approach, N-glycosylation patterns and compositional differences were assessed between tumor and nontumor regions of formalin-fixed clinical ccRCC specimens and tissue microarrays. Regions of normal kidney tissue samples were also evaluated for N-linked glycan-based distinctions between cortex, medullar, glomeruli, and proximal tubule features. Most notable was the proximal tubule localized detection of abundant multiantennary N-glycans with bisecting N-acetylglucosamine and multziple fucose residues. These glycans are absent in ccRCC tissues, while multiple tumor-specific N-glycans were detected with tri- and tetra-antennary structures and varying levels of fucosylation and sialylation. A polycystic kidney disease tissue was also characterized for N-glycan composition, with specific nonfucosylated glycans detected in the cyst fluid regions. Complementary to the imaging mass spectrometry analyses was an assessment of transcriptomic gene array data focused on the fucosyltransferase gene family and other glycosyltransferase genes. The transcript levels of the FUT3 and FUT6 genes responsible for the enzymes that add fucose to N-glycan antennae were significantly decreased in all ccRCC tissues relative to matching nontumor tissues. These striking differences in glycosylation associated with ccRCC could lead to new mechanistic insight into the glycobiology underpinning kidney malignancies and suggest the potential for new therapeutic interventions and diagnostic markers., (© 2019 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of QSOX1 have been shown to suppress tumor growth and invasion in vitro and in vivo , we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as "SBI-183," suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1., (©2019 American Association for Cancer Research.)

Prostate cancer (PCa) is a leading cause of death for men worldwide. Most PCa patients die from metastasis and bone is the most common metastatic site. Three dimensional (3D) porous chitosan-alginate (CA) scaffolds were developed for bone tissue engineering and demonstrated for culture of cancer cells and enrichment of cancer stem cells. However, only a single scaffold composition was studied. Three compositions of 3D porous CA scaffolds (2, 4, and 6 wt%) were used to investigate the effect of scaffold stiffness on PCa cell response with PC-3, C4-2B, and 22Rv1 cell lines. The PC-3 cells formed cell clusters while the C4-2B and 22Rv1 cells formed multicellular spheroids. The three cell lines demonstrated stiffness independent cell growth and expressed phenotypic PCa biomarkers. The osteoblastic PCa lines C4-2B and 22Rv1 mineralized in basal media, while the osteolytic PC-3 line did not, demonstrating that CA scaffold cultures revealed differences in PCa phenotypes. The CA scaffolds are a 3D culture platform that supports PCa growth and phenotypic expression with adjustable scaffold stiffness to mimic stages of metastatic progression. Further investigation of the scaffolds for co-culture of PCa cells with fibroblasts and primary PCa cell culture should be conducted to develop a platform for screening chemotherapies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Introduction: Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers. Progress has been made in treatment of HCC; however, improved outcomes are much needed. The increased metabolic needs of cancer cells underscore the importance of metabolic pathways in cancer cell survival. Lipid metabolism has a role in HCC development; aberrant overexpression of several key enzymes is seen in many solid human tumors. Areas covered: We discuss aberrant lipid metabolism and the promise of multiple targets, in particular related to HCC treatment. We searched PubMed and clinicaltrials.gov for published and unpublished studies from 2000 to 2019. These terms were used: lipids, fatty acid metabolism, lipid metabolism, liver cancer, HCC, de novo fatty acid synthesis, ATP citrate lyase, stearoyl CoA denaturase, fatty acid synthase, acetyl coenzyme A carboxylase, CD147, KLF4, monoglyceride lipase, AMP activated protein kinase. Expert opinion: The importance of dysregulation of fatty acid synthesis in cancer is a growing area of research. HCC demonstrates significant alteration in lipid metabolism, representing great potential as a target for novel therapeutics. Various agents have demonstrated promising anti-neoplastic activity. This strategy deserves further development for improved outcomes.

Purpose: Thyroid cancer cell lines are valuable models but have been neglected in pancancer genomic studies. Moreover, their misidentification has been a significant problem. We aim to provide a validated dataset for thyroid cancer researchers., Experimental Design: We performed next-generation sequencing (NGS) and analyzed the transcriptome of 60 authenticated thyroid cell lines and compared our findings with the known genomic defects in human thyroid cancers., Results: Unsupervised transcriptomic analysis showed that 94% of thyroid cell lines clustered distinctly from other lineages. Thyroid cancer cell line mutations recapitulate those found in primary tumors (e.g., BRAF , RAS , or gene fusions). Mutations in the TERT promoter (83%) and TP53 (71%) were highly prevalent. There were frequent alterations in PTEN , PIK3CA , and of members of the SWI/SNF chromatin remodeling complex, mismatch repair, cell-cycle checkpoint, and histone methyl- and acetyltransferase functional groups. Copy number alterations (CNA) were more prevalent in cell lines derived from advanced versus differentiated cancers, as reported in primary tumors, although the precise CNAs were only partially recapitulated. Transcriptomic analysis showed that all cell lines were profoundly dedifferentiated, regardless of their derivation, making them good models for advanced disease. However, they maintained the BRAF V600E versus RAS-dependent consequences on MAPK transcriptional output, which correlated with differential sensitivity to MEK inhibitors. Paired primary tumor-cell line samples showed high concordance of mutations. Complete loss of p53 function in TP53 heterozygous tumors was the most prominent event selected during in vitro immortalization., Conclusions: This cell line resource will help inform future preclinical studies exploring tumor-specific dependencies., (©2019 American Association for Cancer Research.)

During development of pancreatic cancer, alternatively activated macrophages contribute to fibrogenesis, pancreatic intraepithelial neoplasia (PanIN) lesion growth, and generation of an immunosuppressive environment. Here, we show that the immunomodulatory agent pomalidomide depletes pancreatic lesion areas of alternatively activated macrophage populations. Pomalidomide treatment resulted in downregulation of interferon regulatory factor 4, a transcription factor for M2 macrophage polarization. Pomalidomide-induced absence of alternatively activated macrophages led to a decrease in fibrosis at PanIN lesions and in syngeneic tumors; this was due to generation of an inflammatory, immune-responsive environment with increased expression of IL1α and presence of activated (IFNγ-positive) CD4 + and CD8 + T-cell populations. Our results indicate that pomalidomide could be used to decrease fibrogenesis in pancreatic cancer and may be ideal as a combination treatment with chemotherapeutic drugs or other immunotherapies. SIGNIFICANCE: These findings reveal new insights into how macrophage populations within the pancreatic cancer microenvironment can be modulated, providing the means to turn the microenvironment from immunosuppressive to immune-responsive., (©2019 American Association for Cancer Research.)

Objective: To investigate the molecular underpinnings of thyroid cancer, preclinical cell line models are crucial; however, ∼40% of these have been proven to be either duplicates of existing thyroid lines or even nonthyroid-derived lines or are not derived from humans at all. Therefore, we set out to establish procedures and guidelines that should proactively avoid these problems, which facilitated the creation of criteria to make valid preclinical models for thyroid cancer research., Design: Based on our recommendations, we systematically characterized all new cell lines that we generated by a standardized approach that included (1) determination of human origin, (2) exclusion of lymphoma, (3) DNA fingerprinting and histological comparisons to establish linkage to presumed tissue of origin, (4) examining thyroid differentiation by screening two to three thyroid markers, (5) examination of biological behavior (growth rate, tumorigenicity), and (6) presence of common thyroid cancer genetic changes (TP53, BRAF, PTEN, PIK3CA, RAS, TERT promoter, RET/PTC, PAX8/PPARγ, NF1, and EIF1AX)., Results: We established seven new thyroid cell lines (LAM136, EAM306, SDAR1, SDAR2, JEM493, THJ529, and THJ560) out of 294 primary culture attempts, and 10 patient-derived tumor xenografts (PDTXs; MC-Th-95, MC-Th-374, MC-Th-467, MC-Th-491, MC-Th-493, MC-Th-504, MC-Th-524, MC-Th-529, MC-Th-560, and MC-Th-562) out of 67 attempts. All were successfully validated by our protocols., Conclusions: This standardized approach for cell line and PDTX characterization should prevent (or detect) future cross-contamination and ensure that only valid preclinical models are used for thyroid cancer research.

Systemic treatment is necessary for one third of patients with renal cell carcinoma. No valid biomarker is currently available to tailor personalized therapy. In this study we established a representative panel of patient derived xenograft (PDX) mouse models from patients with renal cell carcinomas and determined serum levels of high mobility group B1 (HMGB1) protein under treatment with sunitinib, pazopanib, sorafenib, axitinib, temsirolimus and bevacizumab. Serum HMGB1 levels were significantly higher in a subset of the PDX collection, which exhibited slower tumor growth during subsequent passages than tumors with low HMGB1 serum levels. Pre-treatment PDX serum HMGB1 levels also correlated with response to systemic treatment: PDX models with high HMGB1 levels predicted response to bevacizumab. Taken together, we provide for the first time evidence that the damage associated molecular pattern biomarker HMGB1 can predict response to systemic treatment with bevacizumab. Our data support the future evaluation of HMGB1 as a predictive biomarker for bevacizumab sensitivity in patients with renal cell carcinoma., Competing Interests: CONFLICTS OF INTEREST JS, KK, AM, ML and ALP are salaried employees of Charles River Discovery, Freiburg, Germany. This employment does not alter our adherence to all the journal policies on sharing data and materials. All experiments were approved by the local ethics committee with written informed consent. The authors declare no competing financial interest.

There is a clear unmet need for novel therapeutic agents for management of primary and secondary brain tumors. Novel therapeutic agents with excellent central nervous system (CNS) penetration and therapeutic activity are urgently needed. EDO-S101 is a novel alkylating and histone deacetylase inhibiting agent created by covalent fusion of bendamustine and vorinostat. We used murine models to perform CNS pharmacokinetic analysis and preclinical therapeutic evaluation of EDO-S101 for CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme. EDO-S101 has excellent CNS penetration of 13.8% and 16.5% by intravenous infusion and bolus administration respectively. It shows promising therapeutic activity against CNS lymphoma, metastatic triple-negative breast cancer of the brain, and glioblastoma multiforme with significant prolongation of survival compared to no-treatment controls. Therapeutic activity was higher with IV infusion compared to IV bolus. It should be evaluated further for therapeutic use in brain tumors., Competing Interests: CONFLICTS OF INTEREST Han W. Tun received a research grant from Mundipharma-EDO GmbH. Thomas Mehrling is an employee of Mundipharma-EDO GmbH.

Triple-negative breast cancer (TNBC) is a heterogeneous disease with poor prognosis that lacks targeted therapies, especially in patients with chemotherapy-resistant disease. Since DNA methylation-induced silencing of tumor suppressors is common in cancer, reversal of promoter DNA hypermethylation by 5-aza-2'-deoxycytidine (decitabine), an FDA-approved DNA methyltransferase (DNMT) inhibitor, has proven effective in treating hematological neoplasms. However, its antitumor effect varies in solid tumors, stressing the importance of identifying biomarkers predictive of therapeutic response. Here, we focused on the identification of biomarkers to select decitabine-sensitive TNBC through increasing our understanding of the mechanism of decitabine action. We showed that protein levels of DNMTs correlated with response to decitabine in patient-derived xenograft (PDX) organoids originating from chemotherapy-sensitive and -resistant TNBCs, suggesting DNMT levels as potential biomarkers of response. Furthermore, all 3 methytransferases, DNMT1, DNMT3A, and DNMT3B, were degraded following low-concentration, long-term decitabine treatment both in vitro and in vivo. The DNMT proteins could be ubiquitinated by the E3 ligase, TNF receptor-associated factor 6 (TRAF6), leading to lysosome-dependent protein degradation. Depletion of TRAF6 blocked decitabine-induced DNMT degradation, conferring resistance to decitabine. Our study suggests a potential mechanism of regulating DNMT protein degradation and DNMT levels as response biomarkers for DNMT inhibitors in TNBCs.

Purpose: The aim of this study was to compare and contrast the expression of all members of the Kallikrein (KLK) family of genes across 15 cancer types and to evaluate their utility as diagnostic and prognostic biomarkers., Results: Severe alterations were found in the expression of different Kallikrein genes across various cancers. Interestingly, renal clear cell and papillary carcinomas have similar kallikrein expression profiles, whereas, chromophobe renal cell carcinoma has a unique expression profile. Several KLK genes have excellent biomarker potential (AUC > 0.90) for chromophobe renal cell carcinoma ( KLK2, KLK3, KLK4, KLK7, KLK15 ), renal papillary carcinoma ( KLK1, KLK6, KLK7 ), clear cell renal cell carcinoma ( KLK1 , KLK6 ), thyroid carcinoma ( KLK2, KLK4, KLK13, KLK15 ) and colon adenocarcinoma ( KLK6, KLK7, KLK8, KLK10 ). Several KLK genes were significantly associated with mortality in clear cell renal cell carcinoma ( KLK2 : HR = 1.69; KLK4 : HR = 1.63; KLK8 : HR = 1.71; KLK10 : HR = 2.12; KLK11 : HR = 1.76; KLK14 : HR = 1.86), papillary renal cell carcinoma ( KLK6 : HR = 3.38, KLK7 : HR = 2.50), urothelial bladder carcinoma ( KLK5 : HR = 1.89, KLK6 : HR = 1.71, KLK8 : HR = 1.60), and hepatocellular carcinoma ( KLK13 : HR = 1.75)., Methods: The RNA-seq gene expression data were downloaded from The Cancer Genome Atlas (TCGA). Statistical analyses, including differential expression analysis, receiver operating characteristic curves and survival analysis (Cox proportional-hazards regression models) were performed., Conclusions: A comprehensive analysis revealed the changes in the expression of different KLK genes associated with specific cancers and highlighted their potential as a diagnostic and prognostic tool., Competing Interests: CONFLICTS OF INTEREST None.

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., Competing Interests: The authors declare no conflict of interest.

Patient-derived tumor xenograft (PDTX) mouse models were used to discover new therapies for naïve and drug resistant BRAF V600E -mutant melanoma. Tumor histology, oncogenic protein expression, and antitumor activity were comparable between patient and PDTX-matched models thereby validating PDTXs as predictive preclinical models of therapeutic response in patients. PDTX models responsive and non-responsive to BRAF/MEK standard of care (SOC) therapy were used to identify efficacious combination therapies. One such combination includes a CDK4/6 inhibitor that blocks cell cycle progression. The rationale for this is that the retinoblastoma protein (pRb) is 95% wildtype in BRAF mutant melanoma. We discovered that 77/77 stage IV metastatic melanoma tissues were positive for inactive phosphorylated pRb (pRb-Ser780). Rb is hyperphosphorylated and inactivated by CDK4/6:cyclin D1 and when restored to its hypophosphorylated active form blocks cell cycle progression. The addition of a CDK4/6 inhibitor to SOC therapy was superior to SOC. Importantly, triple therapy in an upfront treatment and salvage therapy setting provided sustained durable response. We also showed that CDK4/6 blockade resensitized drug resistant melanoma to SOC therapy. Durable response was associated with sustained suppression of pRb-Ser780. Thus, reactivation of pRb may prove to be a clinical biomarker of response and the mechanism responsible for durable response. In light of recent clinical trial data using this triple therapy against BRAF V600E -mutant melanoma, our findings demonstrating superior and prolonged durable response in PDTX models portend use of this therapeutic strategy against naïve and SOC resistant BRAF V600E -mutant metastatic melanoma coupled with pRB-Ser780 as a biomarker of response., Competing Interests: CONFLICTS OF INTEREST LAM, SNM and JAC have received royalties for selected melanoma PDTX models from Charles River Laboratories Inc. for research purposes. SEL, LKD, WFD, DLS, and AJS are employees of Charles River laboratories.

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.

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., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Here we present an innovative computational-based drug discovery strategy, coupled with machine-based learning and functional assessment, for the rational design of novel small molecule inhibitors of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1). Our methods resulted in the discovery of several unique molecules, of which our lead compound SSI-4 demonstrates potent anti-tumor activity, with an excellent pharmacokinetic and toxicology profile. We improve upon key characteristics, including chemoinformatics and absorption/distribution/metabolism/excretion (ADME) toxicity, while driving the IC50 to 0.6 nM in some instances. This approach to drug design can be executed in smaller research settings, applied to a wealth of other targets, and paves a path forward for bringing small-batch based drug programs into the Clinic., Competing Interests: CONFLICTS OF INTEREST There is no conflict of interest.

Context: Recent studies showed that transcription of the MYC gene is driven by the interaction of bromodomain and extraterminal domain (BET) proteins with acetylated histones on chromatin. JQ1, a potent inhibitor that effectively disrupts the interaction of BET proteins with acetylated histones, preferentially suppresses transcription of the MYC gene. We recently reported that JQ1 decreased thyroid tumor growth and improved survival in a mouse model of anaplastic thyroid cancer (ATC) by targeting MYC transcription. The role of MYC in human ATC and whether JQ1 can effectively target MYC as a treatment modality have not been elucidated., Objective: To understand the underlying molecular mechanisms of JQ1, we evaluated its efficacy in human ATC cell lines and xenograft models., Design: We determined the effects of JQ1 on proliferation and invasion in cell lines and xenograft tumors. We identified key regulators critical for JQ1-affected proliferation and invasion of tumor cells., Results: JQ1 markedly inhibited proliferation of four ATC cell lines by suppression of MYC and elevation of p21and p27 to decrease phosphorylated Rb and delay cell cycle progression from the G0/G1 phase to the S phase. JQ1 blocked cell invasion by attenuating epithelial-mesenchymal transition signals. These cell-based studies were further confirmed in xenograft studies in which the size and rate of tumor growth were inhibited by JQ1 via inhibition of p21-cyclin/cyclin-dependent kinase-Rb-E2F signaling., Conclusions: These results suggest targeting of the MYC protein could be a potential treatment modality for human ATC for which effective treatment options are limited., (Copyright © 2017 Endocrine Society)

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Breast cancer patients with residual disease after neoadjuvant chemotherapy (NAC) have increased recurrence risk. Molecular characterization, knowledge of NAC response, and simultaneous generation of patient-derived xenografts (PDXs) may accelerate drug development. However, the feasibility of this approach is unknown., Methods: We conducted a prospective study of 140 breast cancer patients treated with NAC and performed tumor and germline sequencing and generated patient-derived xenografts (PDXs) using core needle biopsies. Chemotherapy response was assessed at surgery., Results: Recurrent "targetable" alterations were not enriched in patients without pathologic complete response (pCR); however, upregulation of steroid receptor signaling and lower pCR rates (16.7%, 1/6) were observed in triple-negative breast cancer (TNBC) patients with luminal androgen receptor (LAR) vs basal subtypes (60.0%, 21/35). Within TNBC, TP53 mutation frequency (75.6%, 31/41) did not differ comparing basal (74.3%, 26/35) and LAR (83.3%, 5/6); however, TP53 stop-gain mutations were more common in basal (22.9%, 8/35) vs LAR (0.0%, 0/6), which was confirmed in The Cancer Genome Atlas and British Columbia data sets. In luminal B tumors, Ki-67 responses were observed in tumors that harbored mutations conferring endocrine resistance ( p53, AKT, and IKBKE ). PDX take rate (27.4%, 31/113) varied according to tumor subtype, and in a patient with progression on NAC, sequencing data informed drug selection (olaparib) with in vivo antitumor activity observed in the primary and resistant (postchemotherapy) PDXs., Conclusions: In this study, we demonstrate the feasibility of tumor sequencing and PDX generation in the NAC setting. "Targetable" alterations were not enriched in chemotherapy-resistant tumors; however, prioritization of drug testing based on sequence data may accelerate drug development., (© The Author, 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com)