A "Prime and Expand" strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy.

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming ("Prime" step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 ("Expand" step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting "Prime and Expand" ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the "Prime and Expand" strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

Simulation-based education for novices: complex learning tasks promote reflective practice.

CONTEXT: Simulated clinical immersion (SCI), in which clinical situations are simulated in a realistic environment, safely and gradually exposes novices to complex problems. Given their limited experience, undergraduate students can potentially be quite overwhelmed by SCI learning tasks, which may result in misleading learning outcomes. Although task complexity should be adapted to the learner's level of expertise, many factors, both intrinsic and extraneous to the learning task, can influence perceived task complexity and its impact on cognitive processes. OBJECTIVES: The purpose of this mixed-methods study was to understand the effects of task complexity on undergraduate pharmacy students' cognitive load, task performance and perception of learning in SCI. METHODS: A total of 167 second-year pharmacy students were randomly assigned to undertake one simple and one complex learning task in SCI consecutively. Participants' cognitive load was measured after each task and debriefing. Task performance and time on task were also assessed. As part of a sequential explanatory design, semi-structured interviews were conducted with students showing maximal variations in intrinsic cognitive load to elucidate their perceptions of learning when dealing with complexity. RESULTS: Although the complex task generated significantly higher cognitive load and time on task than the simple task, performance was high for both tasks. Qualitative results revealed that a lack of clinical experience, an unfamiliar resource in the environment and the constraints inherent to SCI, such as time limitations, hindered the clinical reasoning process and led to poorer self-evaluation of performance. Simple tasks helped students gain more self-confidence, whereas complex tasks further encouraged reflective practice during debriefings. CONCLUSIONS: Although complex tasks in SCI were more cognitively demanding and took longer to execute, students indicated that they learned more from them than they did from simple tasks. Complex tasks constitute an additional challenge in terms of clinical reasoning and thus provide a more valuable learning experience from the student's perspective.

A phase I window, dose escalating and safety trial of metformin in combination with induction chemotherapy in relapsed refractory acute lymphoblastic leukemia: Metformin with induction chemotherapy of vincristine, dexamethasone, PEG-asparaginase, and doxorubicin.

BACKGROUND: Acute lymphoblastic leukemia (ALL) remains a major cause of death in children. AMP-activated protein kinase (AMPK) affects the unfolded protein response (UPR), leading to increased vulnerability to endoplasmic reticulum (ER) stress in ALL cells. In vitro, metformin causes ALL cell death via AMPK-mediated inhibition of the UPR. It was evaluated whether ER stress could be induced in relapsed ALL through a phase I study investigating the safety and feasibility of metformin in combination with relapse induction chemotherapy. PROCEDURE: Metformin was administered twice daily for 28 days in addition to vincristine, dexamethasone, PEG-asparaginase and doxorubicin (VXLD). Dose escalation of metformin was evaluated using a 3+3 design. Pharmacokinetics (PK), pharmacodynamic (PD) evaluation of the AMPK and ER stress/UPR pathways, and treatment response were assessed. RESULTS: Fourteen patients were enrolled; all were evaluable for toxicity. The recommended phase 2 dose (RP2D) was Dose level 2, 1,000 mg/m2 /day. A single dose-limiting toxicity (DLT), hypoglycemia with acidosis, was observed at the RP2D and two DLTs, diarrhea and acidosis, were observed at Dose Level 3. Nine patients were evaluable for response as defined by the protocol, receiving at least 85% of planned metformin doses. Five complete remissions, one partial response, and one stable disease were observed. PD evaluation showed induction of ER stress, activation of AMPK, and inhibition of the UPR. CONCLUSIONS: The VXLD with metformin was tolerable with a RP2D for metformin of 1,000 mg/m2 /day and yielded responses in a heavily pretreated population. ER stress was induced and toxicities attributable to metformin occurred in all dose levels.

Role of the internet as an information resource before anaesthesia consultation: A French prospective multicentre survey.

BACKGROUND: Use of the internet as an information search tool has increased dramatically. Our study assessed preoperative use of the internet by patients to search for information regarding anaesthesia, surgery, pain or outcomes. OBJECTIVE(S): The aim of this study was to test whether patients used the internet prior to surgery and what kinds of information they looked for (anaesthetic technique, pain, adverse events, outcomes and surgery). Correlation between patient age and information sought about surgery from the internet was also explored. DESIGN: A prospective multicentre observational study. SETTING: In total, 14 French private and public institutions from May 2015 to January 2016. PATIENTS: In total, 3161 adult patients scheduled for elective surgery under regional or general anaesthesia. INTERVENTION(S): An anonymous questionnaire was presented to adult patients scheduled for elective surgery under regional or general anaesthesia for completion before the first meeting with the anaesthesiologist. The investigator at each centre completed specific items that the patient could not complete. MAIN OUTCOME MEASURES: We defined the primary endpoint as the number of patients who searched for information about their anaesthesia or surgery on the internet by the time of the their preanaesthetic consultation. RESULTS: Of the 3234 questionnaires distributed, responses were received from 3161 patients. Within this respondent sample, 1304 (45%) were professionally active and 1664 (59%) used the internet at least once per day. Among 3098 (98%) patients who answered the question concerning the primary endpoint, 1506 (48%) had searched the internet for information about their health. In total, 784 (25%) used the internet to find information about their surgery and 113 (3.5%) looked for specific information about anaesthesia. Of the 3161, 52% reported difficulty searching for appropriate information about anaesthesia on the internet. 'Daily use of the web' [odds ratio (OR) 2.0; (95% CI: 1.65 to 2.55) P < 0.001], 'use of the web on mobile devices' [OR 1.24; (95% CI: 1.02 to 1.50) P = 0.02] and 'asking general practitioner or surgeon about information' [OR 1.35; (95% CI: 1.11 to 1.64) P = 0.002] were significantly associated with the primary endpoint. CONCLUSION: The internet was not widely used by patients scheduled for elective surgery to search for information about anaesthesia and surgery in our French multicentre study. TRIAL REGISTRATION: Clinicaltrials.gov identifier: NCT02442609.

Benefits of cardiac coherence combined with medical hypnosis on preoperative anxiety before cancer surgery: the COHEC II study trial protocol.

INTRODUCTION: Preoperative anxiety is a frequent problem that can lead to complications both during anaesthesia and in the postoperative period, especially in oncology. Studies have shown that it can be managed using non-pharmacological approaches, but few works have evaluated psychoeducational programmes. The aim of the COHErence Cardiaque (COHEC) II Study is to evaluate the combination of medical hypnosis (MH) and cardiac coherence (CC) training to manage preoperative anxiety in patients with cancer. METHODS AND ANALYSIS: COHEC II is an ongoing multicentre randomised clinical trial carried out in three French comprehensive cancer centres. In total, 296 patients who will undergo surgery for cancer will be recruited during 18 months and will be randomised in the control arm or the intervention arm. Patients in the intervention arm will follow a daily programme that combines MH and CC, starting 7 days before surgery. The control arm will receive the standard treatment to manage preoperative anxiety. The primary endpoint is the anxiety level on surgery day, measured using a Visual Analogue Scale. Secondary endpoints are patient adherence to the programme, satisfaction and postsurgery recovery quality. ETHICS AND DISSEMINATION: The study protocol was approved by the French Ethics Committee (Comité de Protection des Personnes EST-II) on 24 November 2021 and will be carried out following the good practice guidelines and the Declaration of Helsinki. Results will be published in peer-reviewed journals and presented at conferences. TRIAL REGISTRATION NUMBER: NCT05197972.

Immunotherapeutic approach to reduce senescent cells and alleviate senescence-associated secretory phenotype in mice.

Accumulation of senescent cells (SNCs) with a senescence-associated secretory phenotype (SASP) has been implicated as a major source of chronic sterile inflammation leading to many age-related pathologies. Herein, we provide evidence that a bifunctional immunotherapeutic, HCW9218, with capabilities of neutralizing TGF-ß and stimulating immune cells, can be safely administered systemically to reduce SNCs and alleviate SASP in mice. In the diabetic db/db mouse model, subcutaneous administration of HCW9218 reduced senescent islet ß cells and SASP resulting in improved glucose tolerance, insulin resistance, and aging index. In naturally aged mice, subcutaneous administration of HCW9218 durably reduced the level of SNCs and SASP, leading to lower expression of pro-inflammatory genes in peripheral organs. HCW9218 treatment also reverted the pattern of key regulatory circadian gene expression in aged mice to levels observed in young mice and impacted genes associated with metabolism and fibrosis in the liver. Single-nucleus RNA Sequencing analysis further revealed that HCW9218 treatment differentially changed the transcriptomic landscape of hepatocyte subtypes involving metabolic, signaling, cell-cycle, and senescence-associated pathways in naturally aged mice. Long-term survival studies also showed that HCW9218 treatment improved physical performance without compromising the health span of naturally aged mice. Thus, HCW9218 represents a novel immunotherapeutic approach and a clinically promising new class of senotherapeutic agents targeting cellular senescence-associated diseases.

A Fusion Protein Complex that Combines IL-12, IL-15, and IL-18 Signaling to Induce Memory-Like NK Cells for Cancer Immunotherapy.

Natural killer (NK) cells are a promising cellular therapy for cancer, with challenges in the field including persistence, functional activity, and tumor recognition. Briefly, priming blood NK cells with recombinant human (rh)IL-12, rhIL-15, and rhIL-18 (12/15/18) results in memory-like NK cell differentiation and enhanced responses against cancer. However, the lack of available, scalable Good Manufacturing Process (GMP)-grade reagents required to advance this approach beyond early-phase clinical trials is limiting. To address this challenge, we developed a novel platform centered upon an inert tissue factor scaffold for production of heteromeric fusion protein complexes (HFPC). The first use of this platform combined IL-12, IL-15, and IL-18 receptor engagement (HCW9201), and the second adds CD16 engagement (HCW9207). This unique HFPC expression platform was scalable with equivalent protein quality characteristics in small- and GMP-scale production. HCW9201 and HCW9207 stimulated activation and proliferation signals in NK cells, but HCW9207 had decreased IL-18 receptor signaling. RNA sequencing and multidimensional mass cytometry revealed parallels between HCW9201 and 12/15/18. HCW9201 stimulation improved NK cell metabolic fitness and resulted in the DNA methylation remodeling characteristic of memory-like differentiation. HCW9201 and 12/15/18 primed similar increases in short-term and memory-like NK cell cytotoxicity and IFNγ production against leukemia targets, as well as equivalent control of leukemia in NSG mice. Thus, HFPCs represent a protein engineering approach that solves many problems associated with multisignal receptor engagement on immune cells, and HCW9201-primed NK cells can be advanced as an ideal approach for clinical GMP-grade memory-like NK cell production for cancer therapy.

Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity: role of GATA-4 and GATA-5 in this intermittent process.

Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A- or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.

Inhibition of the NEDD8 conjugation pathway induces calcium-dependent compensatory activation of the pro-survival MEK/ERK pathway in acute lymphoblastic leukemia.

De novo and acquired drug resistance and subsequent relapse remain major challenges in acute lymphoblastic leukemia (ALL). We previously identified that pevonedistat (TAK-924, MLN4924), a first-in-class inhibitor of NEDD8 activating enzyme (NAE), elicits ER stress and has potent in vitro and in vivo efficacy against ALL. However, in pevonedistat-treated ALL cell lines, we found consistent activation of the pro-survival MEK/ERK pathway, which has been associated with relapse and poor outcome in ALL. We uncovered that inhibition of the MEK/ERK pathway in vitro and in vivo sensitized ALL cells to pevonedistat. The observed synergistic apoptotic effect appears to be mediated by inhibition of the MEK/ERK pro-survival cascade leading to de-repression of the pro-apoptotic BIM protein. Mechanistically, Ca2+ influx via the Ca2+-release-activated Ca2+ (CRAC) channel induced protein kinase C ß2 (PKC-ß2) was responsible for activation of the MEK/ERK pathway in pevonedistat-treated ALL cells. Sequestration of Ca2+ using BAPTA-AM or blockage of store-operated Ca2+ entry (SOCE) using BTP-2 both attenuated the compensatory activation of MEK/ERK signaling in pevonedistat-treated ALL cells. Pevonedistat significantly altered the expression of Orai1 and stromal interaction molecule 1 (STIM1), resulting in significantly decreased STIM1 protein levels relative to Orai1. Further, we identified eIF2α as an important post-transcriptional regulator of STIM1, suggesting that pevonedistat-induced eIF2α de-phosphorylation selectively down-regulates translation of STIM1 mRNA. Consequently, our data suggest that pevonedistat potentially activates SOCE and promotes Ca2+ influx leading to activation of the MEK/ERK pathway by altering the stoichiometric Orai1:STIM1 ratio and inducing ER stress in ALL cells.

Effects of a Hypnosis Session Before General Anesthesia on Postoperative Outcomes in Patients Who Underwent Minor Breast Cancer Surgery: The HYPNOSEIN Randomized Clinical Trial.

Importance: Hypnosis is now widespread in medical practice and is emerging as an alternative technique for pain management and anxiety. However, its effects on postoperative outcomes remain unclear. Objective: To evaluate the efficacy of a preoperative hypnosis session for reducing postoperative breast pain in patients who underwent minor breast cancer surgery. Design, Setting, and Participants: The HYPNOSEIN prospective randomized clinical trial was conducted from October 7, 2014, to April 5, 2016. In this multicenter study in France, 150 women scheduled for minor breast cancer surgery were randomized between control and hypnosis arms, and 148 (71 control and 77 hypnosis) were included in the intent-to-treat analysis. Intervention: On the day of surgery, eligible patients were randomly assigned (1:1) to the control arm or the hypnosis arm. Patients (but not the care teams) were blinded to the arm to which they were assigned. A 15-minute hypnosis session before general anesthesia in the operating room was performed in the hypnosis arm. Main Outcomes and Measures: The primary end point was breast pain reduction (by 2 on a visual analog scale), assessed immediately before discharge from the postanesthesia care unit (PACU). Secondary end points were nausea/vomiting, fatigue, comfort/well-being, anxiety, and PACU length of stay, assessed at different times until postoperative day 30. Results: The median patient age was 57 years (range, 33-79 years) in the control arm and 53 years (range, 20-84 years) in the hypnosis arm. Baseline characteristics were similar in the 2 arms. The median duration of the hypnosis session was 6 minutes (range, 2-15 minutes). The use of intraoperative opioids and hypnotics was lower in the hypnosis arm. The mean (SD) breast pain score (range, 0-10) was 1.75 (1.59) in the control arm vs 2.63 (1.62) in the hypnosis arm (P = .004). At PACU discharge and with longer follow-up, no statistically significant difference in breast pain was reported. Fatigue was significantly lower in the hypnosis arm on the evening of surgery (mean [SD] score, 3.81 [2.15] in the control arm vs 2.99 [2.56] in the hypnosis arm; P = .03). The median PACU length of stay was 60 minutes (range, 20-290 minutes) in the control arm vs 46 minutes (range, 5-100 minutes) in the hypnosis arm (P = .002). Exploratory analyses according to patient perception of whether she received hypnosis showed significantly lower fatigue scores in the perceived hypnosis subgroup on the evening of surgery (mean [SD], 4.13 [2.26] for no perceived hypnosis vs 2.97 [2.42] for perceived hypnosis; P = .01). Anxiety was also significantly lower on the evening of surgery in the perceived hypnosis subgroup (mean [SD], 0.75 [1.64] for perceived hypnosis vs 1.67 [2.29] for no perceived hypnosis; P = .03). Conclusions and Relevance: The results of this study do not support a benefit of hypnosis on postoperative breast pain in women undergoing minor breast cancer surgery. However, other outcomes seem to be improved, which needs to be confirmed by further studies. Trial Registration: EudraCT Identifier: 2014-A00681-46 and ClinicalTrials.gov Identifier: NCT03253159.

Cytotoxic effect of 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) on childhood acute lymphoblastic leukemia (ALL) cells: implication for targeted therapy.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy affecting children. Despite significant progress and success in the treatment of ALL, a significant number of children continue to relapse and for them, outcome remains poor. Therefore, the search for novel therapeutic approaches is warranted. The aim of this study was to investigate the AMP activated protein kinase (AMPK) as a potential target in childhood acute lymphoblastic leukemia (ALL) subtypes characterized by non-random translocation signature profiles. We evaluated the effects of the AMPK activator AICAR on cell growth, cell cycle regulators and apoptosis of various childhood ALL cells. RESULTS: We found that treatment with AICAR inhibited cell proliferation, induced cell cycle arrest in G1-phase, and apoptosis in CCRF-CEM (T-ALL), NALM6 (Bp-ALL), REH (Bp-ALL, TEL/AML1) and SupB15 (Bp-ALL, BCR/ABL) cells. These effects were abolished by treatment with the adenosine kinase inhibitor 5'-iodotubericidin prior to addition of AICAR indicating that AICAR's cytotoxicity is mediated through AMPK activation. Moreover, we determined that growth inhibition exerted by AICAR was associated with activation of p38-MAPK and increased expression of the cell cycle regulators p27 and p53. We also demonstrated that AICAR mediated apoptosis through the mitochondrial pathway as revealed by the release of cytochrome C and cleavage of caspase 9. Additionally, AICAR treatment resulted in phosphorylation of Akt suggesting that activation of the PI3K/Akt pathway may represent a compensatory survival mechanism in response to apoptosis and/or cell cycle arrest. Combined treatment with AICAR and the mTOR inhibitor rapamycin resulted in additive anti-proliferative activity ALL cells. CONCLUSION: AICAR-mediated AMPK activation was found to be a proficient cytotoxic agent in ALL cells and the mechanism of its anti-proliferative and apoptotic effect appear to be mediated via activation of p38-MAPK pathway, increased expression of cell cycle inhibitory proteins p27 and p53, and downstream effects on the mTOR pathway, hence exhibiting therapeutic potential as a molecular target for the treatment of childhood ALL. Therefore, activation of AMPK by AICAR represents a novel approach to targeted therapy, and suggests a role for AICAR in combination therapy with inhibitors of the PI3K/Akt/mTOR pathways for the treatment of childhood in ALL.

Calcium influx and DREAM protein are required for GnRH gene expression pulse activity.

Recent evidence using GT1-7 cells indicates that GnRH pulsatility depends on exocytotic-release and gene transcription events. To determine whether calcium or DREAM may play a role in linking these processes, we used an L-type Ca(2+)-blocker (nimodipine) and found that not only GnRH gene expression (GnRH-GE) pulse activity was abolished but also that binding of proteins to OCT1BS-a (essential site for GnRH-GE pulses) was reduced. We further found that only EF-hand forms of DREAM were expressed in GT1-7 and that DREAM was part of the complex binding to OCT1BS-a. Finally, microinjection of DREAM antibody into cells abolished GnRH-GE pulses demonstrating its importance in pulsatility. These results reveal that calcium and DREAM may bridge cytoplasmic and nuclear events enabling temporal coordination of intermittent activity. Expression of DREAM in various cell types coupled with the universal role of calcium raise the possibility that these factors may play similar role in other secretory cells.

Analysis of folylpoly-gamma-glutamate synthetase gene expression in human B-precursor ALL and T-lineage ALL cells.

BACKGROUND: Expression of folylpoly-gamma-glutamate synthetase (FPGS) gene is two- to three-fold higher in B-precursor ALL (Bp- ALL) than in T-lineage ALL (T-ALL) and correlates with intracellular accumulation of methotrexate (MTX) polyglutamates and lymphoblast sensitivity to MTX. In this report, we investigated the molecular regulatory mechanisms directing FPGS gene expression in Bp-ALL and T-ALL cells. METHODS: To determine FPGS transcription rate in Bp-ALL and T-ALL we used nuclear run-on assays. 5'-RACE was used to uncover potential regulatory regions involved in the lineage differences. We developed a luciferase reporter gene assay to investigate FPGS promoter/enhancer activity. To further characterize the FPGS proximal promoter, we determined the role of the putative transcription binding sites NFY and E-box on FPGS expression using luciferase reporter gene assays with substitution mutants and EMSA. RESULTS: FPGS transcription initiation rate was 1.6-fold higher in NALM6 vs. CCRF-CEM cells indicating that differences in transcription rate led to the observed lineage differences in FPGS expression between Bp-ALL and T-ALL blasts. Two major transcripts encoding the mitochondrial/cytosolic and cytosolic isoforms were detected in Bp-ALL (NALM6 and REH) whereas in T-ALL (CCRF-CEM) cells only the mitochondrial/cytosolic transcript was detected. In all DNA fragments examined for promoter/enhancer activity, we measured significantly lower luciferase activity in NALM6 vs. CCRF-CEM cells, suggesting the need for additional yet unidentified regulatory elements in Bp-ALL. Finally, we determined that the putative transcription factor binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineage. CONCLUSION: We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, suggesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory elements, transcription initiation rates, and mRNA processing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance or inducing apoptosis in leukemic cells.

The NEDD8-activating enzyme inhibitor pevonedistat activates the eIF2α and mTOR pathways inducing UPR-mediated cell death in acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related death in children, and cure rates for adults remain dismal. Further, effective treatment strategies for relapsed/refractory ALL remain elusive. We previously uncovered that ALL cells are prone to apoptosis via endoplasmic reticulum (ER) stress/unfolded protein response (UPR)-mediated mechanisms. We investigated the antineoplastic activity of pevonedistat®, a novel NEDD8-activating enzyme inhibitor that targets E3 cullin-RING ligases (CRLs) dependent proteasomal protein degradation, in ALL. Herein, we report that pevonedistat induces apoptosis in ALL cells by dysregulating the translational machinery leading to induction of proteotoxic/ER stress and UPR-mediated cell death. Mechanistically, pevonedistat led to P-eIF2a dephosphorylation causing atypical proteotoxic/ER stress from failure to halt protein translation via the UPR and upregulation of mTOR/p70S6K. Additional studies revealed that pevonedistat re-balanced the homeostasis of pro- and anti-apoptotic proteins to favor cell death through altered expression and/or activity of Mcl-1, NOXA, and BIM, suggesting that pevonedistat has a "priming" effect on ALL by altering the apoptotic threshold through modulation of Mcl-1 activity. Further, we demonstrated that pevonedistat synergizes with selected anti-leukemic agents in vitro, and prolongs survival of NSG mice engrafted with ALL cells, lending support for the use of pevonedistat as part of a multi-agent approach.

Pulses of prolactin promoter activity depend on a noncanonical E-box that can bind the circadian proteins CLOCK and BMAL1.

Recent findings from our laboratory and those of others demonstrated that prolactin gene expression (PRL-GE) oscillates in single living mammotropes, but little information is available on the molecular processes that contribute to this phenomenon. To elucidate the source of this activity, we generated a series of constructs containing decreasing lengths of the PRL promoter fused to a luciferase reporter gene. These constructs were injected into single cells and assayed for photonic activity. We found pulse activity with all plasmids tested, even with the smallest promoter fragment of 331 bp. Sequence analysis of this fragment identified two potential E-boxes (elements known to bind CLOCK and BMAL1 circadian proteins). Furthermore, RT-PCR of PRL cells (pituitary, MMQ, and GH(3)) revealed expression of clock and bmal1 as well as five other clock genes (per1, per2, cry1, cry2, and tim), suggesting that the circadian system may function in PRL cells. Next, we mutated the core sequences of both E-boxes within the 2.5-kb PRL promoter and found that only mutation of the E-box133 completely abolished PRL-GE pulses. EMSAs revealed that CLOCK and BMAL1 were able to bind to the E-box133 site in vitro. Our results demonstrate that PRL-GE pulses are dependent on a specific E-box binding site in the PRL promoter. Moreover, the indication that CLOCK/BMAL1 can bind to this site suggests that these circadian proteins, either alone or in conjunction with other factors, may regulate intermittent PRL promoter activity in mammotropes, perhaps by acting as a temporal switch for the on/off expression of PRL.

Identification of a novel OCT1 binding site that is necessary for the elaboration of pulses of rat GnRH promoter activity.

Recent evidence from our laboratory demonstrated that the OCT1 protein was necessary for GnRH gene promoter pulse activity through its interaction with a specific OCT1 binding site (OCT1BS-a, -1,774/-1,781). In light of the importance of this POU homeoprotein in pulsatile function, we focused on two other highly conserved OCT1 sites within this region, OCT1BS-b (-1,694/-1,701, previously AT-b), and OCT1BS-c (-1,569/-1,562). Mutagenesis of these sites revealed that alteration of OCT1BS-c, but not OCT1BS-b, virtually abolished gene expression pulses in GT1-7 cells. EMSAs confirmed that OCT1 can bind to both sites. Taken together, our findings demonstrate clearly that more than one Oct1 binding site is necessary for GnRH promoter pulses. Moreover, the lack of an influence observed with OCT1BS-b on pulse activity indicates that OCT1 action is not general to all OCT1 sites, but specific to certain octamer sequences in the NSE region of the GnRH promoter.

Episodic activation of the rat GnRH promoter: role of the homeoprotein oct-1.

Recent reports demonstrate that the rat GnRH promoter is activated in an episodic fashion in immortalized GnRH neurons, but little information is available on molecular processes that contribute to this phenomenon. In this study, we dissected the regions of the rat GnRH promoter that mediate these effects by testing a series of 5' deletion luciferase reporter constructs on the pattern of photonic emissions from single, living GT1-7 GnRH neuronal cells. Deletion analysis revealed that the region -2012/-1597 that contains the neuron-specific enhancer (NSE) was required for the elaboration of pulses of GnRH promoter activity. The importance of this region was supported by observations that episodic reporter activity could be transferred to a neutral nonpulsatile promoter (Rous sarcoma virus, RSV(180)). Immunoneutralization of Oct-1 as well as mutation of an octamer binding site located at -1787/-1783 (AT-a site) blocked the pulsatile GnRH promoter activity in GT1-7 neuronal cells. Taken together, our findings indicate that episodic GnRH gene expression is a promoter-dependent phenomenon, which is mediated by Oct-1 interaction with regulatory elements in the NSE region.

Mcl-1 downregulation leads to the heightened sensitivity exhibited by BCR-ABL positive ALL to induction of energy and ER-stress.

BCR-ABL positive (+) acute lymphoblastic leukemia (ALL) accounts for ∼30% of cases of ALL. We recently demonstrated that 2-deoxy-d-glucose (2-DG), a dual energy (glycolysis inhibition) and ER-stress (N-linked-glycosylation inhibition) inducer, leads to cell death in ALL via ER-stress/UPR-mediated apoptosis. Among ALL subtypes, BCR-ABL+ ALL cells exhibited the highest sensitivity to 2-DG suggesting BCR-ABL expression may be linked to this increased vulnerability. To confirm the role of BCR-ABL, we constructed a NALM6/BCR-ABL stable cell line and found significant increase in 2-DG-induced apoptosis compared to control. We found that Mcl-1 was downregulated by agents inducing ER-stress and Mcl-1 levels correlated with ALL sensitivity. In addition, we showed that Mcl-1 expression is positively regulated by the MEK/ERK pathway, dependent on BCR-ABL, and further downregulated by combining ER-stressors with TKIs. We determined that energy/ER stressors led to translational repression of Mcl-1 via the AMPK/mTOR and UPR/PERK/eIF2α pathways. Taken together, our data indicate that BCR-ABL+ ALL exhibits heightened sensitivity to induction of energy and ER-stress through inhibition of the MEK/ERK pathway, and translational repression of Mcl-1 expression via AMPK/mTOR and UPR/PERK/eIF2α pathways. This study supports further consideration of strategies combining energy/ER-stress inducers with BCR-ABL TKIs for future clinical translation in BCR-ABL+ ALL patients.

PRL gene expression in individual living mammotropes displays distinct functional pulses that oscillate in a noncircadian temporal pattern.

PRL gene expression in the anterior pituitary has been the focus of intensive investigation for many years, but very little information is available on the actual dynamics by which this process occurs in individual mammotrope cells. Here, we used single cell bioluminescent imaging microscopy and a recently refined reporter gene strategy to measure PRL promoter-driven gene expression (PRL-GE) in individual living primary mammotropes. Using this approach we report a new phenomenon involving repetitive on/off gene expression bursts that occurred in a distinctly noncircadian oscillatory pattern. Furthermore, we demonstrate a functional basis for these gene expression oscillations, inasmuch as PRL-GE pulses were sensitive to calcium-dependent modulation, which we show arose exclusively as changes in the shape of individual pulse episodes. Our results provide the first clear evidence that PRL-GE, in its homologous cell environment, displays oscillatory bursts of activity. Moreover, they strongly support the idea that these discrete on/off bursts of activity serve as an important determinant of the timing and level of PRL-GE under both basal and stimulated conditions.

Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines.

BACKGROUND: We describe an alternative method to determine mRNA half-life (t1/2) based on the Real-Time RT-PCR procedure. This approach was evaluated by using the beta-actin gene as a reference molecule for measuring of mRNA stability. RESULTS: Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGree&ncircledR; fluorescent dye with the VersaFluor Fluorometer System. One &mgr;g of total RNA was reverse transcribed and used as template for the amplification of a region of the beta-actin gene (231 bp). To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, beta-actin mRNAs were quantified by Real-Time RT-PCR using the SYB&RcircledR; Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the beta-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h). CONCLUSIONS: We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that beta-actin mRNA half-life can be affected by the cellular growth rate.