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Introduction: Teclistamab, the first approved B-cell maturation antigen-directed bispecific antibody for treatment of triple-class exposed relapsed/refractory multiple myeloma, demonstrated deep, durable responses with a manageable safety profile in the pivotal MajesTEC-1 cohort (NCT03145181/NCT04557098). Efficacy, safety, and pharmacokinetics from the MajesTEC-1 China cohort are reported., Methods: Patients received teclistamab 1.5 mg/kg subcutaneously weekly after step-up dosing; patients could switch to less frequent dosing with continued response., Results: In the China cohort (N = 26; median age, 66 years; median prior lines of therapy, 5) 15-month median follow-up, overall response rates, very good partial response or better, and complete response or better (≥CR) were 76.9%, 76.9%, and 57.7%, respectively. Median time to first response and ≥CR were 1.4 and 6.3 months, respectively; among patients with ≥CR and have available MRD samples, MRD negativity was achieved in 14/15 (93.3%) patients. Median duration of response, progression-free survival, and overall survival were not reached; 12-month duration of response, progression-free survival, and overall survival rates were 78.5%, 68.0%, and 83.5%, respectively. The safety profile was consistent with the pivotal cohort. Although infections occurred in 96.2% of patients, incidence decreased over time with six patients experiencing infections for >12 to 18 months. There were no discontinuations because of adverse events and no dose reductions. Ten patients switched to less frequent dosing. Teclistamab serum concentrations were consistent with the pivotal cohort, with a slightly lower mean pharmacokinetics profile., Conclusions: Teclistamab demonstrated efficacy and safety profiles in the China cohort consistent with the pivotal cohort, supporting teclistamab as a promising treatment option for triple-class exposed relapsed/refractory multiple myeloma in China., (© 2024 The Author(s). Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Abstract: Teclistamab is a B-cell maturation antigen (BCMA)-directed bispecific antibody approved for the treatment of patients with triple-class exposed relapsed/refractory multiple myeloma (R/RMM). In the phase 1/2 MajesTEC-1 study, a cohort of patients who had prior BCMA-targeted therapy (antibody-drug conjugate [ADC] or chimeric antigen receptor T-cell [CAR-T] therapy) was enrolled to explore teclistamab in patients previously exposed to anti-BCMA treatment. At a median follow-up of 28.0 months (range, 0.7-31.1), 40 patients with prior BCMA-targeted therapy had received subcutaneous 1.5 mg/kg weekly teclistamab. The median prior lines of treatment was 6 (range, 3-14). Prior anti-BCMA therapy included ADC (n = 29), CAR-T (n = 15), or both (n = 4). The overall response rate was 52.5%; 47.5% of patients achieved very good partial response or better, and 30.0% achieved complete response or better. The median duration of response was 14.8 months, the median progression-free survival was 4.5 months, and the median overall survival was 15.5 months. The most common treatment-emergent adverse events (TEAEs) were neutropenia, infections, cytokine release syndrome, and anemia; cytopenias and infections were the most common grade ≥3 TEAEs. Infections occurred in 28 patients (70.0%; maximum grade 3/4, n = 13 [32.5%]; grade 5, n = 4 [10%]). Before starting teclistamab, baseline BCMA expression and immune characteristics were unaffected by prior anti-BCMA treatment. The MajesTEC-1 trial cohort C results demonstrate favorable efficacy and safety of teclistamab in patients with heavily pretreated R/RMM and prior anti-BCMA treatment. This trial was registered at www.ClinicalTrials.gov as #NCT03145181 and #NCT04557098., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Abstract: Teclistamab, an off-the-shelf B-cell maturation antigen (BCMA) × CD3 bispecific antibody that mediates T-cell activation and subsequent lysis of BCMA-expressing myeloma cells, is approved for the treatment of patients with relapsed/refractory multiple myeloma (R/RMM). As a T-cell redirection therapy, clinical outcomes with teclistamab may be influenced by patient immune fitness and tumor antigen expression. We correlated tumor characteristics and baseline immune profiles with clinical response and disease burden in patients with R/RMM from the pivotal phase 1/2 MajesTEC-1 study, focusing on patients treated with 1.5 mg/kg of teclistamab (N = 165). Peripheral blood samples were collected at screening, and bone marrow samples were collected at screening and cycle 3. Better clinical outcomes to teclistamab correlated with higher baseline total T-cell counts in the periphery. In addition, responders (partial response or better) had a lower proportion of immunosuppressive regulatory T cells (Tregs), T cells expressing coinhibitory receptors (CD38, PD-1, and PD-1/TIM-3), and soluble BCMA and a T-cell profile suggestive of a more cytolytic potential, compared with nonresponders. Neither frequency of baseline bone marrow BCMA expression nor BCMA-receptor density was associated with clinical response to teclistamab. Improved progression-free survival was observed in patients with a lower frequency of T cells expressing exhaustion markers and immunosuppressive Tregs. Overall, response to teclistamab was associated with baseline immune fitness; nonresponders had immune profiles suggestive of immune suppression and T-cell dysfunction. These findings illustrate the importance of the contribution of the immune landscape to T-cell redirection therapy response. This trial was registered at www.ClinicalTrials.gov as #NCT03145181/NCT04557098., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Purpose: Bispecific antibodies (BsAb) directed against B-cell maturation antigen (teclistamab) or the orphan G protein-coupled receptor GPRC5D (talquetamab) induce deep and durable responses in heavily pretreated patients with multiple myeloma. However, mechanisms underlying primary and acquired resistance remain poorly understood., Experimental Design: The anti-multiple myeloma activity of teclistamab and talquetamab was evaluated in bone marrow (BM) samples from patients with multiple myeloma. T-cell phenotype and function were assessed in BM/peripheral blood samples obtained from patients with multiple myeloma who were treated with these BsAb., Results: In ex vivo killing assays with 41 BM samples from BsAb-naive patients with multiple myeloma, teclistamab- and talquetamab-mediated multiple myeloma lysis was strongly correlated (r = 0.73, P < 0.0001). Both BsAb exhibited poor activity in samples with high regulatory T-cell (Treg) numbers and a low T-cell/multiple myeloma cell ratio. Furthermore, comprehensive phenotyping of BM samples derived from patients treated with teclistamab or talquetamab revealed that high frequencies of PD-1+ CD4+ T cells, CTLA4+ CD4+ T cells, and CD38+ CD4+ T cells were associated with primary resistance. Although this lack of response was linked to a modest increase in the expression of inhibitory receptors, increasing T-cell/multiple myeloma cell ratios by adding extra T cells enhanced sensitivity to BsAb. Further, treatment with BsAb resulted in an increased proportion of T cells expressing exhaustion markers (PD-1, TIGIT, and TIM-3), which was accompanied by reduced T-cell proliferative potential and cytokine secretion, as well as impaired antitumor efficacy in ex vivo experiments., Conclusions: Primary resistance is characterized by a low T-cell/multiple myeloma cell ratio and Treg-driven immunosuppression, whereas reduced T-cell fitness due to continuous BsAb-mediated T-cell activation may contribute to the development of acquired resistance., (©2024 American Association for Cancer Research.)

Natural killer/T cell lymphoma (NKTCL) is a highly aggressive, heterogeneous non-Hodgkin lymphoma resulting from malignant proliferation of cytotoxic natural killer (NK) or T cells. Previous studies demonstrated variable expression of CD38 on NKTCL tumors. Daratumumab, a human IgGκ monoclonal antibody targeting CD38 with a direct on-tumor and immunomodulatory mechanism of action, was hypothesized to be a novel therapeutic option for patients with relapsed or refractory (R/R) NKTCL. In the phase 2 NKT2001 study (ClinicalTrials.gov Identifier: NCT02927925) assessing the safety and efficacy of daratumumab, a suboptimal overall response rate was seen in R/R NKTCL patients. One patient, whose tumors did not express CD38, responded to treatment, suggesting that the immunomodulatory activities of daratumumab may be sufficient to confer clinical benefit. To understand the suboptimal response rate and short duration of response, we investigated the immune profile of NKTCL patients from NKT2001 in the context of daratumumab anti-tumor activity. Tumor tissue and whole blood were, respectively, analyzed for CD38 expression and patient immune landscapes, which were assessed via cytometry by time-of-flight (CyTOF), multiparameter flow cytometry (MPFC), clonal sequencing, and plasma Epstein-Barr virus (EBV)-DNA level measurements. Changes observed in the immune profiles of NKTCL patients from NKT2001, including differences in B and T cell populations between responders and nonresponders, suggest that modulation of the immune environment is crucial for daratumumab anti-tumor activities in NKTCL. In conclusion, these findings highlight that the clinical benefit of daratumumab in NKTCL may be enriched by B/T cell-related biomarkers., (© 2024. The Author(s).)

Abstract: Teclistamab and other B-cell maturation antigen (BCMA)-targeting bispecific antibodies (BsAbs) have substantial activity in patients with heavily pretreated multiple myeloma (MM) but are associated with a high rate of infections. BCMA is also expressed on normal plasma cells and mature B cells, which are essential for the generation of a humoral immune response. The aim of this study was to improve the understanding of the impact of BCMA-targeting BsAbs on humoral immunity. The impact of teclistamab on polyclonal immunoglobulins and B cell counts was evaluated in patients with MM who received once-weekly teclistamab 1.5 mg/kg subcutaneously. Vaccination responses were assessed in a subset of patients. Teclistamabinduced rapid depletion of peripheral blood B cells in patients with MM and eliminated normal plasma cells in ex vivo assays. In addition, teclistamab reduced the levels of polyclonal immunoglobulins (immunoglobulin G [IgG], IgA, IgE, and IgM), without recovery over time while receiving teclistamab therapy. Furthermore, response to vaccines against Streptococcus pneumoniae, Haemophilus influenzae type B, and severe acute respiratory syndrome coronavirus 2 was severely impaired in patients treated with teclistamab compared with vaccination responses observed in patients with newly diagnosed MM or relapsed/refractory MM. Intravenous immunoglobulin (IVIG) use was associated with a significantly lower risk of serious infections among patients treated with teclistamab (cumulative incidence of infections at 6 months: 5.3% with IVIG vs 54.8% with observation only [P < .001]). In conclusion, our data show severe defects in humoral immunity induced by teclistamab, the impact of which can be mitigated by the use of immunoglobulin supplementation. This trial was registered at www.ClinicalTrials.gov as #NCT04557098., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The CD38-targeting antibody daratumumab has marked activity in multiple myeloma (MM). Natural killer (NK) cells play an important role during daratumumab therapy by mediating antibody-dependent cellular cytotoxicity via their FcγRIII receptor (CD16), but they are also rapidly decreased following initiation of daratumumab treatment. We characterized the NK cell phenotype at baseline and during daratumumab monotherapy by flow cytometry and cytometry by time of flight to assess its impact on response and development of resistance (DARA-ATRA study; NCT02751255). At baseline, nonresponding patients had a significantly lower proportion of CD16 + and granzyme B + NK cells, and higher frequency of TIM-3 + and HLA-DR + NK cells, consistent with a more activated/exhausted phenotype. These NK cell characteristics were also predictive of inferior progression-free survival and overall survival. Upon initiation of daratumumab treatment, NK cells were rapidly depleted. Persisting NK cells exhibited an activated and exhausted phenotype with reduced expression of CD16 and granzyme B, and increased expression of TIM-3 and HLA-DR. We observed that addition of healthy donor-derived purified NK cells to BM samples from patients with either primary or acquired daratumumab-resistance improved daratumumab-mediated MM cell killing. In conclusion, NK cell dysfunction plays a role in primary and acquired daratumumab resistance. This study supports the clinical evaluation of daratumumab combined with adoptive transfer of NK cells., Competing Interests: MCM has a consultancy or advisory role for Gilead Sciences, BMS, Alnylam, Janssen Cilag, Takeda, and Servier; all paid to employer, hospitality from Celgene. MDL serves in advisory boards for Roche, Janssen and Abbvia. AB receives honoraria from Celgene, Janssen, Amgen, Takeda, and Sanofi. MJK has received research support from Kite/Gilead, and honoraria from Kite/Gilead, Novartis, BMS/Celgene, Takeda, Roche, and Miltenyi Biotec (all paid to institution). MK, TC, YA, RIV, TS, GV, DCS, LvS, and ER are employed by Janssen. PS has received honoraria from Amgen, BMS, Celgene, Janssen, Karyopharm, Takeda, and receives research funding from Amgen, Celgene, Janssen, Karyopharm, SkylineDx, Takeda. SZ has received research funding from Celgene, Takeda, Janssen, and serves in advisory boards for Janssen, Takeda, BMS, Oncopeptides and Sanofi, all paid to institution. TM has received research support from Janssen Pharmaceuticals, Takeda, Genmab, Novartis, and ONK Therapeutics. NWCJvdD has received research support from Janssen Pharmaceuticals, AMGEN, Celgene, Novartis, Cellectis, and BMS, and serves in advisory boards for Janssen Pharmaceuticals, AMGEN, Celgene, BMS, Takeda, Roche, Novartis, Bayer, Adaptive, and Servier. All the other authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

Purpose: Preclinical characterization of cetrelimab (JNJ-63723283), a fully humanized immunoglobulin G4 kappa monoclonal antibody targeting programmed cell death protein-1 (PD-1), in human cancer models., Methods: Cetrelimab was generated by phage panning against human and cynomolgus monkey (cyno) PD-1 extracellular domains (ECDs) and affinity maturation. Binding to primate and rodent PD-1 ECDs, transfected and endogenous cell-surface PD-1, and inhibition of ligand binding were measured. In vitro activity was evaluated using cytomegalovirus recall, mixed lymphocyte reaction, staphylococcal enterotoxin B stimulation, and Jurkat-PD-1 nuclear factor of activated T cell reporter assays. In vivo activity was assessed using human PD-1 knock-in mice implanted with MC38 tumors and a lung patient-derived xenograft (PDX) model (LG1306) using CD34 cord-blood-humanized NSG mice. Pharmacodynamics, toxicokinetics, and safety were assessed in cynos following single and/or repeat intravenous dosing., Results: Cetrelimab showed high affinity binding to human (1.72 nM) and cyno (0.90 nM) PD-1 and blocked binding of programmed death-ligand 1 (PD-L1; inhibitory concentration [IC] 111.7 ng/mL) and PD-L2 (IC 138.6 ng/mL). Cetrelimab dose-dependently increased T cell-mediated cytokine production and stimulated cytokine expression. Cetrelimab 10 mg/kg reduced mean MC38 tumor volume in PD-1 knock-in mice at Day 21 (P < 0.0001) versus control. In a PDX lung model, 10 mg/kg cetrelimab (every 5 days for six cycles) increased frequency of peripheral T cells and reduced (P < 0.05) mean tumor volume versus control. Activity was consistent with that of established PD-1 inhibitors. Cetrelimab dosing was well tolerated in cynos and mean drug exposure increase was dose-dependent., Conclusion: Cetrelimab potently inhibits PD-1 in vitro and in vivo, supporting its clinical evaluation., (© 2022. The Author(s).)

Cell surface expression levels of GPRC5D, an orphan G protein-coupled receptor, are significantly higher on multiple myeloma (MM) cells, compared with normal plasma cells or other immune cells, which renders it a promising target for immunotherapeutic strategies. The novel GPRC5D-targeting T-cell redirecting bispecific antibody, talquetamab, effectively kills GPRC5D+ MM cell lines in the presence of T cells from both healthy donors or heavily pretreated MM patients. In addition, talquetamab has potent anti-MM activity in bone marrow (BM) samples from 45 patients, including those with high-risk cytogenetic aberrations. There was no difference in talquetamab-mediated killing of MM cells from newly diagnosed, daratumumab-naïve relapsed/refractory (median of 3 prior therapies), and daratumumab-refractory (median of 6 prior therapies) MM patients. Tumor cell lysis was accompanied by T-cell activation and degranulation, as well as production of pro-inflammatory cytokines. High levels of GPRC5D and high effector:target ratio were associated with improved talquetamab-mediated lysis of MM cells, whereas an increased proportion of T cells expressing PD-1 or HLA-DR, and elevated regulatory T-cell (Treg) counts were associated with suboptimal killing. In cell line experiments, addition of Tregs to effector cells decreased MM cell lysis. Direct contact with bone marrow stromal cells also impaired the efficacy of talquetamab. Combination therapy with daratumumab or pomalidomide enhanced talquetamab-mediated lysis of primary MM cells in an additive fashion. In conclusion, we show that the GPRC5D-targeting T-cell redirecting bispecific antibody talquetamab is a promising novel antimyeloma agent. These results provide the preclinical rationale for ongoing studies with talquetamab in relapsed/refractory MM., (© 2021 by The American Society of Hematology.)

PD1 blockade to reinvigorate T cells has become part of standard of care for patients with NSCLC across disease stages. However, the majority of patients still do not respond. One potential mechanism of resistance is increased expression of other checkpoint inhibitory molecules on T cells leading to their suppression; however, this phenomenon has not been well studied in tumor-reactive, human T cells. The purpose of this study was to evaluate this compensatory mechanism in a novel model using human effector T cells infiltrating and reactive against human lung cancer. Immunodeficient mice with flank tumors established from a human lung cancer cell line expressing the NYESO1 antigen were treated with activated human T cells expressing a TCR reactive to NYESO1 (Ly95) with or without anti-PD1 alone and with combinations of anti-PD1 plus anti-TIM3 or anti-TIGIT. A month later, the effect on tumor growth and the phenotype and ex vivo function of the TILs were analyzed. Anti-PD1 and Ly95 T cells led to greater tumor control than Ly95 T cells alone; however, tumors continued to grow. The ex-vivo function of PD1-blocked Ly95 TILs was suppressed and was associated with increased T cell expression of TIM3/TIGIT. Administering combinatorial blockade of PD1+ TIM3 or PD1+ TIGIT with Ly95 T cells led to greater tumor control than blocking PD1 alone. In our model, PD1 blockade was suboptimally therapeutic alone. The effect of TIM3 and TIGIT was upregulated on T cells in response to PD1 blockade and anti-tumor activity could be enhanced when these inhibitory receptors were also blocked with antibodies in combination with anti-PD1 therapy., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2 K660N /p53 mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti-PD-1 alone was ineffective, the erdafitinib and anti-PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRAS G12C -mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti-PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti-PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival., (©2019 American Association for Cancer Research.)

Efficacious antitumor immune responses must overcome multiple suppressive mechanisms in the tumor microenvironment to control cancer progression. In this study, we demonstrate that dual targeting of suppressive myeloid populations by inhibiting CSF-1/CSF-1R signaling and activation of antigen-presenting cells with agonist anti-CD40 treatment confers superior antitumor efficacy and increased survival compared with monotherapy treatment in preclinical tumor models. Concurrent CSF-1R blockade and CD40 agonism lead to profound changes in the composition of immune infiltrates, causing an overall decrease in immunosuppressive cells and a shift toward a more inflammatory milieu. Anti-CD40/anti-CSF-1R-treated tumors contain decreased tumor-associated macrophages and Foxp3 + regulatory T cells. This combination approach increases maturation and differentiation of proinflammatory macrophages and dendritic cells and also drives potent priming of effector T cells in draining lymph nodes. As a result, tumor-infiltrating effector T cells exhibit improved responses to tumor antigen rechallenge. These studies show that combining therapeutic approaches may simultaneously remove inhibitory immune populations and sustain endogenous antitumor immune responses to successfully impair cancer progression. Cancer Immunol Res; 5(12); 1109-21. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Genomic imprinting governs allele-specific gene expression in an epigenetically heritable manner. The characterization of histone modifications at imprinted gene loci is incomplete, and whether specific histone marks determine transcription or are dependent on it is not understood. Using chromatin immunoprecipitations, we examined in multiple cell types and in an allele-specific manner the active and repressive histone marks of several imprinted loci, including H19, KvDMR1, Snrpn promoter/exon 1, and IG-DMR imprinting control regions. Expressed alleles are enriched for specific actively modified histones, including H3 di- and trimethylated at Lys4 and acetylated histones H3 and H4, while their silent counterparts are associated with repressive marks such as H3 trimethylated at Lys9 alone or in combination with H3 trimethylated at Lys27 and H4/H2A symmetrically dimethylated at Arg3. At H19, allele-specific histone modifications occur throughout the entire locus, including nontranscribed regions such as the differentially methylated domain (DMD) as well as sequences in the H19 gene body that are not differentially methylated. Significantly, the presence of active marks at H19 depends on transcriptional activity and occurs even in the absence of the DMD. These findings suggest that histone modifications are dependent on the transcriptional status of imprinted alleles and illuminate epigenetic mechanisms of genomic imprinting.

Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

X chromosome inactivation (XCI) is the phenomenon through which one of the two X chromosomes in female mammals is silenced to achieve dosage compensation with males. XCI is a highly complex, tightly controlled and developmentally regulated process. The mouse undergoes two forms of XCI: imprinted, which occurs in all cells of the preimplantation embryo and in the extraembryonic lineage, and random, which occurs in somatic cells after implantation. This review presents results and hypotheses that have recently been proposed concerning important aspects of both imprinted and random XCI in mice. We focus on how imprinted XCI occurs during preimplantation development, including a brief discussion of the debate as to when silencing initiates. We also discuss regulation of random XCI, focusing on the requirement for Tsix antisense transcription through the Xist locus, on the regulation of Xist chromatin structure by Tsix and on the effect of Tsix regulatory elements on choice and counting. Finally, we review exciting new data revealing that X chromosomes co-localize during random XCI. To conclude, we highlight other aspects of X-linked gene regulation that make it a suitable model for epigenetics at work.

Preimplantation development is a period of dynamic epigenetic change that begins with remodeling of egg and sperm genomes, and ends with implantation. During this time, parental-specific imprinting marks are maintained to direct appropriate imprinted gene expression. We previously demonstrated that H19 imprinting could be lost during preimplantation development under certain culture conditions. To define the lability of genomic imprints during this dynamic period and to determine whether loss of imprinting continues at later stages of development, imprinted gene expression and methylation were examined after in vitro preimplantation culture. Following culture in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed and undermethylated. However, only a subset of individual cultured blastocysts (approximately 65%) exhibited biallelic expression, while others maintained imprinted H19 expression. Loss of H19 imprinting persisted in mid-gestation conceptuses. Placental tissues displayed activation of the normally silent allele for H19, Ascl2, Snrpn, Peg3 and Xist while in the embryo proper imprinted expression for the most part was preserved. Loss of imprinted expression was associated with a decrease in methylation at the H19 and Snrpn imprinting control regions. These results indicate that tissues of trophectoderm origin are unable to restore genomic imprints and suggest that mechanisms that safeguard imprinting might be more robust in the embryo than in the placenta.

The regulation of H19 and Igf2 imprinting and expression depends on common elements. Using comparative analysis between human and mouse, we identified conserved regions 3' of the H19 transcription unit, including the H19/Igf2 endodermal enhancers and elements within a 4.2-kb domain between the H19 transcription unit and the enhancers. Transgene experiments implicate these elements in imprinting regulation. To establish whether they are required at the endogenous locus, first we replaced the endodermal enhancers with the alpha-fetoprotein endodermal enhancers (H19Afp). Second, we deleted the 4.2-kb region (H19delta4.2). Our analysis revealed that H19 and Igf2 imprinting and tissue-specific expression were maintained for both mutations, except for a slight reduction in paternal Igf2 expression from the H19Afp allele in liver. These results demonstrate that the H19 insulator can interact with heterologous enhancers to imprint Igf2. Furthermore, for H19, chromatin context or additional sequences possibly compensate for loss of conserved 3' elements., (Copyright 2004 Elsevier Inc.)

Cloning by somatic cell nuclear transfer requires that epigenetic information possessed by the donor nucleus be reprogrammed to an embryonic state. Little is known, however, about this remodeling process, including when it occurs, its efficiency, and how well epigenetic markings characteristic of normal development are maintained. Examining the fate of epigenetic information associated with imprinted genes during clonal development offers one means of addressing these questions. We examined transcript abundance, allele specificity of imprinted gene expression, and parental allele-specific DNA methylation in cloned mouse blastocysts. Striking disruptions were seen in total transcript abundance and allele specificity of expression for five imprinted genes. Only 4% of clones recapitulated a blastocyst mode of expression for all five genes. Cloned embryos also exhibited extensive loss of allele-specific DNA methylation at the imprinting control regions of the H19 and Snprn genes. Thus, epigenetic errors arise very early in clonal development in the majority of embryos, indicating that reprogramming is inefficient and that some epigenetic information may be lost.

An intriguing characteristic of imprinted genes is that they often cluster in large chromosomal domains, raising the possibility that gene-specific and domain-specific mechanisms regulate imprinting. Several common features emerged from comparative analysis of four imprinted domains in mice and humans: (a) Certain genes appear to be imprinted by secondary events, possibly indicating a lack of gene-specific imprinting marks; (b) some genes appear to resist silencing, predicting the presence of cis-elements that oppose domain-specific imprinting control; (c) the nature of the imprinting mark remains incompletely understood. In addition, common silencing mechanisms are employed by the various imprinting domains, including silencer elements that nucleate and propagate a silent chromatin state, insulator elements that prevent promoter-enhancer interactions when hypomethylated on one parental allele, and antisense RNAs that function in silencing the overlapping sense gene and more distantly located genes. These commonalities are reminiscent of the behavior of genes subjected to, and the mechanisms employed in, dosage compensation.