A major challenge in stem-cell mediated regenerative medicine is the development of defined culture systems for the maintenance of clinical-grade human embryonic stem (hES) cells. Using a feedback system control scheme, we identified a unique combination of three small molecule inhibitors that enables the maintenance of hES cells on a fibronectin-coated surface through single cell passaging. After 20 passages, the undifferentiated state of the hES cells was confirmed by OCT4, SSEA4 and NANOG expressions, while their pluripotent potential and genetic integrity were demonstrated by teratoma formation and normal karyotype, respectively. Our study attests to the power of feedback system control scheme in quickly pinpointing optimal conditions for desired biological activities and provides a chemically defined, scalable and single cell passaging culture system for hES cells.
Purpose To carry out an integrative profile of human pancreatic ductal adenocarcinoma (PDAC) to identify prognosis-significant genes and their related pathways. Experimental Design A concordant survival-based whole genome in silico array analysis of DNA copy number, and mRNA and miRNA expression in 25 early-stage PDAC was carried out. A novel composite score simultaneously integrated gene expression with regulatory mechanisms to identify the signature genes with the most levels of prognosis-significant evidence. The predominant signaling pathways were determined via a pathway-based approach. Independent patient cohorts (n = 148 and 42) were then used as in vitro validation of the array findings. Results The composite score identified 171 genes in which expressions were able to define two prognosis subgroups (P = 3.8e-5). Eighty-eight percent (151 of 171) of the genes were regulated by prognosis-significant miRNAs. The phosphoinositide 3-kinase/AKT pathway and SRC signaling were densely populated by prognosis-significant genes and driven by genomic amplification of SRC and miRNA regulation of p85α and CBL. On tissue microarray validation (n = 148), p85α protein expression was associated with improved survival for all patients (P = 0.02), and activated P-SRC (Y418) was associated shorter survival for patients with low-grade histology tumors (P = 0.04). Interacting P-SRC and p85α revealed that they define two distinct PDAC patient subgroups (P = 0.0066). Furthering the importance of these pathways, CBL protein expression was associated with improved survival (P = 0.03) on a separate cohort (n = 42). Conclusions These pathways and related genes may represent putative clinical biomarkers and possible targets of individualized therapy in the distinct patient subgroups they define.
New therapies for late stage and castration resistant prostate cancer (CRPC) depend on defining unique properties and pathways of cell sub-populations capable of sustaining the net growth of the cancer. One of the best enrichment schemes for isolating the putative stem/progenitor cell from the murine prostate gland is Lin-;Sca1+;CD49fhi (LSChi), which results in a more than 10-fold enrichment for in vitro sphere-forming activity. We have shown previously that the LSChi subpopulation is both necessary and sufficient for cancer initiation in the Pten-null prostate cancer model. To further improve this enrichment scheme, we searched for cell surface molecules upregulated upon castration of murine prostate and identified CD166 as a candidate gene. CD166 encodes a cell surface molecule that can further enrich sphere-forming activity of WT LSChi and Pten null LSChi. Importantly, CD166 could enrich sphere-forming ability of benign primary human prostate cells in vitro and induce the formation of tubule-like structures in vivo. CD166 expression is upregulated in human prostate cancers, especially CRPC samples. Although genetic deletion of murine CD166 in the Pten null prostate cancer model does not interfere with sphere formation or block prostate cancer progression and CRPC development, the presence of CD166 on prostate stem/progenitors and castration resistant sub-populations suggest that it is a cell surface molecule with the potential for targeted delivery of human prostate cancer therapeutics.
The prognosis for patients with pancreatic ductal adenocarcinoma (PDAC) is dismal. Although gemcitabine (GEM) is the standard chemotherapeutic agent for adjuvant therapy of resectable PDAC, recurrent disease is observed in an alarming number of GEM-treated patients. Regardless of the adjuvant therapy, the vast majority of patients treated with chemotherapy after surgical resection show tumor recurrence. A better understanding of the molecular mechanisms that contribute to chemoresistance would aid the development of more effective treatment strategies. GRP78 is an endoplasmic reticulum (ER) chaperone protein that primarily resides in the lumen of the ER and is the master regulator of the unfolded protein response (UPR). Here, we report that expression of GRP78 is significantly higher in GEM-resistant PDAC compared to GEM-sensitive PDAC patient samples. We show that GRP78 induces chemoresistance in PDAC cells. Our results also show that knockdown of GRP78 reduces chemoresistance in PDAC. Finally, we found that IT-139, a ruthenium-based anticancer drug, can overcome GRP78-mediated chemoresistance. In vitro, IT-139 restores sensitivity to cytotoxic drugs in drug-resistant PDAC cells and induces twice as much cell death in combination treatment compared with GEM alone. In vivo, a single weekly IT-139 treatment in combination with GEM caused a 35% increase in median survival and a 25% increase in overall survival compared to GEM alone. Collectively, our data show that GRP78 expression promotes chemoresistance in PDAC and therapeutic strategies, blocking the activity of GRP78 increases the efficacy of currently available therapies.
7508 Background: In this open label phase 1 dose escalation study, safety, tolerability, pharmacokinetics, pharmacodynamics and preliminary efficacy of AMG 330 were evaluated in patients (pts) with R/R AML (NCT#02520427). Methods: AMG 330 was evaluated as a continuous IV (cIV) infusion using a 3+3 design. Response was assessed per revised IWG criteria. Each cycle (2–4 weeks duration) was followed by an infusion-free interval. Eligible pts were ≥18 y/o with > 5% blasts in bone marrow and ≥1 line/s of prior therapy. Results: As of December 10, 2019, 55 pts (median age, 58.0 [18.0–80.0] years) were enrolled in 16 cohorts. AMG 330 was administered on 4 schedules (0–3 dose steps) prior to the target dose (TD, 0.5–720 µg/day). Dose steps were implemented in the dose schedule design based on the adverse event (AE) profile. Across all schedules, 55 (100%) pts reported treatment-emergent AEs (any grade). AMG 330–related AEs reported in 49/55 (89%) pts included cytokine release syndrome (CRS; 67%; ≥ grade 3 in 13%), (60%) and nausea (20%) as the most frequent AEs. CRS was reversible and occurred in a dose/schedule-dependent manner mostly within the first 24 hours of administration of triggering AMG 330 dose. The frequency and severity of CRS correlated with the dose level and leukemic burden at baseline. AMG 330 exhibited dose-dependent increase in steady state exposures over the studied dose range with clinical PK profile consistent with cIV administration. Eight of 42 evaluable pts responded: 3 complete remissions (CR; including 1 CR with negative measurable residual disease reported after data snapshot), 4 CR with incomplete hematologic recovery, and 1 morphologic leukemia free state. Seven responders who achieved CR/CRi received a TD equal or above the minimal efficacious dose of 120 μg/day. Among analyzed CR/CRi responders, 4/6 (67%) had adverse cytogenetic risk profile, 3/6 (50%) had ≥4 lines of prior therapy and all had relapsed disease. Responders had higher AMG 330 exposures and 3 responders treated with ≥600 μg/day TD remain in CR/CRi: 1 patient for > 5 months after cycle 1, 1 patient bridged to hematopoietic stem cell transplant after cycle 4 and 1 patient is in cycle 3. Preliminary response assessment showed a correlation with lower tumor burden at baseline with a trend towards higher CD8+ lymphocyte count and E:T ratio. Conclusions: AMG 330 dosed up to 720 μg/day provided early evidence of acceptable safety profile, drug tolerability and anti-leukemic activity, and supports further dose escalation. Clinical trial information: NCT02520427 .
Background: AMG 673 is a novel half-life extended (HLE) BiTE® (bispecific T-cell engager) construct that binds both CD33 and CD3 and is genetically fused to the N-terminus of a single-chain IgG Fc region, thereby potentially increasing the half-life of the molecule. AMG 673 redirects T cells toward CD33+ cells, with the induced proximity leading to T-cell‒mediated cytotoxicity against acute myeloid leukemia (AML) blasts. Anti-AML activity of other CD33/CD3 bispecific T-cell engager molecules has been previously reported (Blood, 2018, 132, 25; Blood, 2018, 132, 1455). The objectives of this ongoing study are to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of AMG 673 in adult patients aged ≥18 years with relapsed/refractory (R/R) AML. Methods: This is an ongoing first-in-human, open-label, phase 1, sequential dose escalation study (NCT03224819). AMG 673 was administered as two, short, and intermittent intravenous (IV) infusions during a 14-day cycle in adult patients with R/R AML. Patients received treatment cycles of AMG 673 until disease progression or unacceptable toxicities. T-cell activation, cytokine, and AMG 673 levels in patients' blood were evaluated by validated assays. Results were summarized descriptively by the dosing cohorts and potential associations between PK, PD, safety, and preliminary efficacy were evaluated. Results: As of June 14, 2019, 30 patients had enrolled in 10 cohorts and were treated with AMG 673 (dose range, 0.05-72 μg IV per dose). The median age was 67.5 (range: 25.0-84.0) years; 20/30 (67%) patients had received ≥4 prior anti-AML treatments, baseline myelosuppression at study entry was common (grade ≥3 neutropenia 21/30 [70%], thrombocytopenia 25/30 [83%], leukopenia 14/30 [47%]), and 7/30 (23%) patients had undergone hematopoietic stem cell transplant (HSCT) before enrolling in the study. Patients received a median of 1.5 (range: 1.0-6.0) cycles of AMG 673; 27/30 (90%) patients discontinued treatment due to disease progression (n=21), patient request (n=2), protocol-specified criteria (n=2), or adverse events (AEs; n=2). A total of 3 patients were still receiving AMG 673 at the time of data analysis. The most common treatment-related AE was cytokine release syndrome (CRS) reported in 15/30 (50%) patients (grade 1, n=6; grade 2, n=5; grade 3, n=4; no grade 4 CRS). Treatment-related serious AEs were reported in 11/30 (37%) patients, and 15/30 (50%) patients experienced treatment-related AEs of grade ≥3, with the most common being abnormal hepatic enzymes (n=5, 17%), CRS (n=4, 13%), leukopenia (n=4, 13%), thrombocytopenia (n=2, 7%), and febrile neutropenia (n=2, 7%). Two deaths, unrelated to AMG 673, were reported on days 19 and 28 after the last dose. Assessment of bone marrow in treated patients showed a decrease in blasts in 12/27 (44%) evaluable patients, of which 6 experienced ≥50% reduction in blasts compared with baseline (Figure 1). One patient achieved complete remission with incomplete hematologic recovery (CRi) with 85% reduction in bone marrow blasts at a dose of 36 µg. Dose-related increases in Cmax and AUC were observed following AMG 673 infusions. Preliminary half-life estimates for AMG 673 were longer than those observed for canonical CD33-specific BiTE® molecule with short half-lives. Upregulation of T-cell activation markers CD25 and CD69 on T-cell subsets and cytokine release post-infusion were observed at higher doses. Preliminary associations between AMG 673 exposures, T-cell activation, safety, and clinical response have been evaluated. Conclusions: Preliminary data of AMG 673 dosed up to 72 µg provide early evidence of the molecule's acceptable safety profile, drug tolerability, and anti-leukemic activity. An association was observed between PK/PD relationships that were consistent with the biological activity of AMG 673. These preliminary results support further dose escalation of the AMG 673 HLE BiTE® molecule in patients with R/R AML. Disclosures Subklewe: AMGEN: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Morphosys: Research Funding; Janssen: Consultancy; Celgene: Consultancy, Honoraria; Miltenyi: Research Funding; Oxford Biotherapeutics: Research Funding; Pfizer: Consultancy, Honoraria. Stein:Celgene: Speakers Bureau; Stemline: Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Walter:Amgen: Consultancy; Amphivena Therapeutics: Consultancy, Equity Ownership; Aptevo Therapeutics: Consultancy, Research Funding; Argenx BVBA: Consultancy; Astellas: Consultancy; BioLineRx: Consultancy; BiVictriX: Consultancy; Seattle Genetics: Research Funding; Boehringer Ingelheim: Consultancy; Boston Biomedical: Consultancy; Covagen: Consultancy; Daiichi Sankyo: Consultancy; Agios: Consultancy; Race Oncology: Consultancy; Jazz Pharmaceuticals: Consultancy; Kite Pharma: Consultancy; Pfizer: Consultancy, Research Funding; New Link Genetics: Consultancy. Wei:Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau. Ritchie:Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy; BMS: Research Funding; Takeda: Research Funding; Beigene: Research Funding; Imago: Research Funding; Novartis: Honoraria; Sanofi: Honoraria. Vachhani:AbbVie: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Astellas: Speakers Bureau. Dai:Amgen: Employment, Equity Ownership. Hindoyan:Amgen Inc.: Employment, Other: stock ownership. Agarwal:Amgen: Employment, Equity Ownership; AbbVie: Equity Ownership. Anderson:Amgen Inc.: Employment, Equity Ownership. Khaldoyanidi:Amgen: Employment, Equity Ownership; BMS: Equity Ownership. Ravandi:Macrogenix: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Selvita: Research Funding; Cyclacel LTD: Research Funding.
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