Maintenance of Paternal Methylation and Repression of the Imprinted H19 Gene Requires MBD3

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

Teclistamab impairs humoral immunity in patients with heavily pretreated myeloma:importance of immunoglobulin supplementation

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 onceweekly 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 &lt; .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.</p

Teclistamab impairs humoral immunity in patients with heavily pretreated myeloma:importance of immunoglobulin supplementation

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 onceweekly 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 &lt; .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.</p

Genetic subtypes of smoldering multiple myeloma are associated with distinct pathogenic phenotypes and clinical outcomes

Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with significant heterogeneity in disease progression. Existing clinical models of progression risk do not fully capture this heterogeneity. Here we integrate 42 genetic alterations from 214 SMM patients using unsupervised binary matrix factorization (BMF) clustering and identify six distinct genetic subtypes. These subtypes are differentially associated with established MM-related RNA signatures, oncogenic and immune transcriptional profiles, and evolving clinical biomarkers. Three genetic subtypes are associated with increased risk of progression to active MM in both the primary and validation cohorts, indicating they can be used to better predict high and low-risk patients within the currently used clinical risk stratification models

NK Cell Phenotype Is Associated With Response and Resistance to Daratumumab in Relapsed/Refractory Multiple Myeloma

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

Repression of the Arf tumor suppressor by E2F3 is required for normal cell cycle kinetics

Tumor development is dependent upon the inactivation of two key tumor-suppressor networks, p16(Ink4a)–cycD/cdk4–pRB–E2F and p19(Arf)–mdm2–p53, that regulate cellular proliferation and the tumor surveillance response. These networks are known to intersect with one another, but the mechanisms are poorly understood. Here, we show that E2F directly participates in the transcriptional control of Arf in both normal and transformed cells. This occurs in a manner that is significantly different from the regulation of classic E2F-responsive targets. In wild-type mouse embryonic fibroblasts (MEFs), the Arf promoter is occupied by E2F3 and not other E2F family members. In quiescent cells, this role is largely fulfilled by E2F3b, an E2F3 isoform whose function was previously undetermined. E2f3 loss is sufficient to derepress Arf, triggering activation of p53 and expression of p21(Cip1). Thus, E2F3 is a key repressor of the p19(Arf)–p53 pathway in normal cells. Consistent with this notion, Arf mutation suppresses the activation of p53 and p21(Cip1) in E2f3-deficient MEFs. Arf loss also rescues the known cell cycle re-entry defect of E2f3(-/-) cells, and this correlates with restoration of appropriate activation of classic E2F-responsive genes. Our data also demonstrate a direct role for E2F in the oncogenic activation of Arf. Specifically, we observe recruitment of the endogenous activating E2Fs, E2F1, and E2F3a, to the Arf promoter. Thus, distinct E2F complexes directly contribute to the normal repression and oncogenic activation of Arf. We propose that monitoring of E2F levels and/or activity is a key component of Arf's ability to respond to inappropriate, but not normal cellular proliferation

The Transcriptional Status but Not the Imprinting Control Region Determines Allele-Specific Histone Modifications at the Imprinted H19 Locus▿

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