Transcriptional and Epigenetic Consequences of DMSO Treatment on HepaRG Cells

Dimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPARα whereas genes not affected by this addition are regulated by HNF1α, HNF4α, and PPARα. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation

Limited efficacy of APRIL CAR in patients with multiple myeloma indicate challenges in the use of natural ligands for CAR T-cell therapy

BACKGROUND: We used a proliferating ligand (APRIL) to construct a ligand-based third generation chimeric antigen receptor (CAR) able to target two myeloma antigens, B-cell maturation antigen (BCMA) and transmembrane activator and CAML interactor. METHODS: The APRIL CAR was evaluated in a Phase 1 clinical trial (NCT03287804, AUTO2) in patients with relapsed, refractory multiple myeloma. Eleven patients received 13 doses, the first 15×106 CARs, and subsequent patients received 75,225,600 and 900×106 CARs in a 3+3 escalation design. RESULTS: The APRIL CAR was well tolerated. Five (45.5%) patients developed Grade 1 cytokine release syndrome and there was no neurotoxicity. However, responses were only observed in 45.5% patients (1×very good partial response, 3×partial response, 1×minimal response). Exploring the mechanistic basis for poor responses, we then compared the APRIL CAR to two other BCMA CARs in a series of in vitro assays, observing reduced interleukin-2 secretion and lack of sustained tumor control by APRIL CAR regardless of transduction method or co-stimulatory domain. There was also impaired interferon signaling of APRIL CAR and no evidence of autoactivation. Thus focusing on APRIL itself, we confirmed similar affinity to BCMA and protein stability in comparison to BCMA CAR binders but reduced binding by cell-expressed APRIL to soluble BCMA and reduced avidity to tumor cells. This indicated either suboptimal folding or stability of membrane-bound APRIL attenuating CAR activation. CONCLUSIONS: The APRIL CAR was well tolerated, but the clinical responses observed in AUTO2 were disappointing. Subsequently, when comparing the APRIL CAR to other BCMA CARs, we observed in vitro functional deficiencies due to reduced target binding by cell-expressed ligand

Potentiel de différenciation et de transdifférenciation de cellules d'hépatome humain HepaRG (étude des mécanismes associés)

Le carcinome hépatocellulaire est l'une des tumeurs malignes les plus répandues. La caractérisation de la lignée d'hépatome humain HepaRG nous a permis de démontrer i) sa bipotence liée à la différenciation en hépatocytes ou cellules biliaires ii) le potentiel de transdifférenciation de ces cellules différenciées iii) l'autorenouvellement des hépatocytes différenciés. Les communications cellulaires sont importantes dans ces processus via les voies de signalisation Notch et Wnt/ -caténine corrélées à l'expression de facteurs de transcription hépatocytaires. Ces résultats suggèrent que la croissance tumorale est associée à la prolifération des hépatocytes et que la transdifférenciation est un mécanisme susceptible d'alimenter le pool de progéniteurs hépatiques présents au sein de tumeurs différenciées. L'étude du métabolisme des lipides révèle que HepaRG exprime des enzymes fonctionnelles impliquées dans la transformation des acides gras, tout comme les hépatocytes humains normaux.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Targeting the polyamine transport system with benzazepine- and azepine-polyamine conjugates.

International audienceThe polyamine transport system (PTS) whose activity is up-regulated in cancer cells is an attractive target for drug design. Two heterocyclic (azepine and benzazepine) systems were conjugated to various polyamine moieties through an amidine bound to afford 18 compounds which were evaluated for their affinity for the PTS and their ability to use the PTS for cell delivery. Structure-activity relationship studies and lead optimization afforded two attractive PTS targeting compounds. The azepine-spermidine conjugate 14 is a very selective substrate of the PTS that may serve as a vector for radioelements used for diagnoses or therapeutics in nuclear medicine. The nitrobenzazepine-spermine conjugate 28 is a very powerful PTS inhibitor with very low intrinsic cytotoxicity, able to prevent the growth of polyamine depleted cells in presence of exogenous polyamines

CYP4F3B expression is associated with differentiation of HepaRG human hepatocytes and unaffected by fatty acid overload.

International audienceFatty acid microsomal ω-oxidation involves cytochrome P450 enzymes. Some of them belonging to the CYP4F3 family are mainly expressed in the liver, making this organ a major player in energy homeostasis and lipid metabolism. To study this important regulation pathway, we used HepaRG cells, which gradually undergo a complete differentiation process. Even at the early stage of the differentiation process, CYP4F3B generated by alternative splicing of the CYP4F3 gene represented the prevalent isoform in HepaRG cells as in the liver. Its increasing expression associated with hepatocyte differentiation status suggested a hepatic-specific control of this isoform. As in liver microsomes, the catalytic hydroxylation of the CYP4F3B substrate [1-¹⁴C]Z9(10)-epoxystearic acid led to major production of 18-hydroxy-9(10)-epoxystearic acid. When treated with saturated, monounsaturated, or polyunsaturated fatty acids, CYP4F3B and CYP4A11 expression remained unchanged whereas CYP4F2 and CYP4F12 expression was transiently up-regulated. A 24-h exposure of differentiated HepaRG cells to various polyunsaturated fatty acids and derivatives induced microvesicular steatosis; down-regulation of lipid metabolism gene regulators such as sterol regulatory element-binding protein-1c, fatty acid synthase, peroxisome proliferator-activated receptor γ (PPARγ), PPARα, and decreased expression of glucose-dependent metabolism genes, which could limit de novo lipogenesis. Docosahexaenoic acid seemed to be the most effective compound. These results suggest that a PPARα-independent pathway could participate to limit lipogenesis and emphasize the role of hepatocytes in the fatty acid ω-hydroxylation pathway. They also give insights on the use of HepaRG hepatocytes to open new avenues of investigations on factors mediating the lipid metabolic pathway and finding new hypolipidemic molecules

Transdifferentiation of hepatocyte-like cells from the human hepatoma HepaRG cell line through bipotent progenitor.

International audienceHepatic tumors, exhibiting mature hepatocytes and undifferentiated cells merging with cholangiocyte and hepatocyte phenotypes, are frequently described. The mechanisms by which they occur remain unclear. We report differentiation and transdifferentiation behaviors of human HepaRG cells isolated from a differentiated tumor developed consecutively to chronic HCV infection. We demonstrate that, in vitro, proliferating HepaRG cells differentiate toward hepatocyte-like and biliary-like cells at confluence. If hepatocyte-like cells are selectively isolated and cultured at high cell density, they proliferate and preserve their differentiation status. However, when plated at low density, they transdifferentiate into hepatocytic and biliary lineages through a bipotent progenitor. In accordance, transplantation of either undifferentiated or differentiated HepaRG cells in uPA/SCID mouse damaged liver gives rise mainly to functional human hepatocytes infiltrating mouse parenchyma. Analysis of the differentiation/transdifferentiation process reveals that: (1) the reversible differentiation fate of HepaRG cells is related to the absence of p21(CIP1) and p53 accumulation in differentiated cells; (2) HepaRG bipotent progenitors express the main markers of in vivo hepatic progenitors, and that cell differentiation process is linked to loss of their expression; (3) early and transient changes of beta-catenin localization and HNF3beta expression are correlated to Notch3 upregulation during hepatobiliary commitment of HepaRG cells. CONCLUSION: Our results demonstrate the great plasticity of transformed hepatic progenitor cells and suggest that the transdifferentiation process could supply the pool of hepatic progenitor cells. Moreover, they highlight possible mechanisms by which transdifferentiation and proliferation of unipotent hepatocytes might cooperate in the development of mixed and differentiated tumors

Transcriptional and epigenetic consequences of DMSO treatment on HepaRG cells

International audienceDimethyl sulfoxide (DMSO) is used to sustain or favor hepatocyte differentiation in vitro. Thus, DMSO is used in the differentiation protocol of the HepaRG cells that present the closest drug-metabolizing enzyme activities to primary human hepatocytes in culture. The aim of our study is to clarify its influence on liver-specific gene expression. For that purpose, we performed a large-scale analysis (gene expression and histone modification) to determine the global role of DMSO exposure during the differentiation process of the HepaRG cells. The addition of DMSO drives the upregulation of genes mainly regulated by PXR and PPAR alpha whereas genes not affected by this addition are regulated by HNF1 alpha, HNF4 alpha, and PPAR alpha. DMSO-differentiated-HepaRG cells show a differential expression for genes regulated by histone acetylation, while differentiated-HepaRG cells without DMSO show gene signatures associated with histone deacetylases. In addition, we observed an interplay between cytoskeleton organization and EMC remodeling with hepatocyte maturation

Safety and Preliminary Efficacy of Sabestomig (AZD7789), an Anti-PD-1 and Anti-TIM-3 Bispecific Antibody, in Patients with Relapsed or Refractory Classical Hodgkin Lymphoma Previously Treated with Anti-PD-(L)1 Therapy

Background Immunotherapies targeting the PD-1/PD-L1 pathway have been shown to induce high response rates in patients with relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). However, most responses are partial and not durable. T cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) is implicated as a resistance mechanism following anti-PD-1 therapy, and increased TIM-3 expression has been observed on T cells of patients with cHL post anti-PD-1 therapy. Therefore, we hypothesized that dual targeting of PD-1 and TIM-3 may reinvigorate immune responses and lead to more durable antitumor activity. Sabestomig is a monovalent, bispecific, humanized IgG1 monoclonal antibody that binds PD-1 and a unique epitope in the TIM-3 IgV domain without blocking phosphatidylserine binding, eliciting differential functionality. In preclinical mouse models, sabestomig was more effective at inhibiting growth of solid tumors than treatment targeting only PD-1, and sequential sabestomig after anti-PD-1 therapy increased antitumor responses. Here we report preliminary data from the ongoing dose escalation portion of a Phase I/II, open-label, multicenter study to assess the safety and preliminary efficacy of sabestomig in patients with r/r cHL (NCT05216835). Methods Eligible patients are aged ≥16 years with r/r cHL, an Eastern Cooperative Oncology Group performance status 0-1, at least one positron emission tomography-avid measurable lesion according to modified Lugano Criteria, and exposure to at least 2 prior lines of systemic therapy including a minimum of 3 cycles of an anti-PD-(L)1-based therapy. Sabestomig is intravenously infused over 1 hour following pretreatment with diphenhydramine and acetaminophen every 3 weeks, with planned doses ranging from 2 to 2000 mg across 8 cohorts (A1-A8); cohorts A1 to A4 (2, 7, 22.5 or 75 mg) follow an accelerated titration design with a single patient treated at each dose level, while cohorts A5 to A8 (225-2000 mg) follow a modified toxicity probability interval-2 algorithm. The primary endpoint of the dose escalation part of the study is safety, including dose-limiting toxicities (DLTs). Secondary endpoints include efficacy, pharmacokinetics, and immunogenicity. Data cutoffs were May 17, 2023 for safety and June 20, 2023 for efficacy. Results As of May 17, 2023, 14 patients were treated across the first 6 cohorts (A1-A6; 2-750 mg). They were predominantly male (64.3%) with Stage IV disease (85.7%) and a median age of 41.5 years (range, 25-77). Patients received a median of 6 prior lines of therapy (range, 3-13). Baseline disease characteristics are summarized in Table 1. Four patients received sabestomig 2-75 mg in cohorts A1-A4 (n=1 each), 5 patients received 225 mg in cohort A5, and 5 patients received 750 mg in cohort A6. Median duration of exposure to sabestomig was 8.9 weeks (range, 3.0-34.3) with a median of 3 cycles received (range 1-12). Sabestomig was well tolerated with no Grade ≥3 treatment-related adverse events (AEs) (Table 2). One Grade ≥3 AE occurred, which was fatal (sepsis secondary to gastric ulcer rupture, not related to sabestomig but deemed a DLT by the safety review committee). No immune-mediated AEs were reported, and no AEs led to treatment discontinuation. At the safety data cutoff, treatment was ongoing for 6 patients (42.9%). As of June 20, 2023, 11 patients had their first disease assessment and 4/11 had an objective response based on modified Lugano Criteria: 1 patient in cohort A3 (22.5 mg, a partial response [PR]) and all 3 patients who had their first disease assessment in cohort A6 (750 mg, 2 CRs and 1 PR). Two of these 4 responders (both in cohort A6) had experienced a best overall response of progressive disease during prior anti-PD-1 therapy; the other two initially had a response (1 CR [cohort A6] and 1 PR [cohort A3]) but subsequently progressed on anti-PD-1 therapy. Sustained PD-1 receptor occupancy (≥90%) was observed in peripheral blood at doses ≥225 mg. Conclusions Sabestomig was well tolerated with a manageable safety profile. Early efficacy data at 750 mg is encouraging with objective responses in 3/3 patients who had their first disease assessment, including those who were anti-PD-1 refractory or who relapsed on treatment. Updated data will be presented from ongoing and subsequent patients treated during dose escalation with doses of up to 2000 mg