Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctions.

Genes encoding immunoglobulin heavy chains (Igh) are assembled by rearrangement of variable (V(H)), diversity (D(H)) and joining (J(H)) gene segments. Three critical constraints govern V(H) recombination. These include timing (V(H) recombination follows D(H) recombination), precision (V(H) gene segments recombine only to DJ(H) junctions) and allele specificity (V(H) recombination is restricted to DJ(H)-recombined alleles). Here we provide a model for these universal features of V(H) recombination. Analyses of DJ(H)-recombined alleles showed that DJ(H) junctions were selectively epigenetically marked, became nuclease sensitive and bound RAG recombinase proteins, which thereby permitted D(H)-associated recombination signal sequences to initiate the second step of Igh gene assembly. We propose that V(H) recombination is precise, because these changes did not extend to germline D(H) segments located 5' of the DJ(H) junction.

Risk of myeloproliferative neoplasms among U.S. Veterans from Korean, Vietnam, and Persian Gulf War eras.

The Promise to Address Comprehensive Toxics (PACT) Act expanded U.S. Veterans' health care and benefits for conditions linked to service-connected exposures (e.g., Burn Pits, Agent Orange). However, myeloproliferative neoplasms (MPN) are not recognized as presumptive conditions for Veterans exposed to these toxic substances. This study evaluated the development of MPN among U.S. Veterans from the Korean, Vietnam, and Persian Gulf War eras. This retrospective cohort study included 65 425 Korean War era Veterans; 211 927 Vietnam War era Veterans; and 214 007 Persian Gulf War era Veterans from January 1, 2006, to January 26, 2023. Veterans with MPN, thrombosis, bleeding, and cardiovascular risk factors were identified through ICD-9 and -10 codes. Veterans from the Persian Gulf War era had the highest risk of developing MPN compared with Veterans from the Korean and Vietnam War eras, hazard ratio (HR) 4.92, 95% confidence interval (CI) 4.20-5.75 and HR 2.49, 95% CI 2.20-2.82, both p < .0001, respectively. Vietnam War era Veterans also had a higher risk of MPN development compared with Korean War era Veterans, HR 1.97, 95% CI 1.77-2.21, p < .0001. Persian Gulf War era Veterans were diagnosed with MPN at an earlier age, had higher risks of thrombosis and bleeding, and had lower survival rates compared with Korean War and Vietnam War era Veterans. This study reinforces evidence that environmental and occupational hazards increase the risk of clonal myeloid disorders and related complications, impacting overall survival with MPN. Limitations include the inability to confirm clonality and fully verify deployment and exposure status.

The prevalence and clinical significance of HER2 expression in prostate adenocarcinoma.

AIMS: Abnormalities in HER2 are well-established oncogenic drivers and are approved therapeutic targets in various malignancies. Prior studies evaluating HER2 expression in prostate cancer (PCa) have yielded variable results. Most of these studies used immunohistochemistry scoring systems based on breast cancer data. The goal of this study was to determine the prevalence and clinical significance of HER2 expression using a scoring system that better reflects the HER2 staining pattern observed in PCa. METHODS: We randomly selected similar numbers of localized low risk (AJCC stage I), locally advanced (AJCC stages II & III), and metastatic (AJCC stage IV) PCa patients treated at the DC VA Medical Center between 2000 and 2022. Among them, we included patients who had sufficient PCa tissue samples and clinical information required for this analysis. Two experienced pathologists independently scored HER2 expression (Ventana Pathway anti-HER2) according to a modified gastric cancer HER2 scoring system. RESULTS: Out of the 231 patients included, 85 % self-identified as Black. 58.9 % of patients expressed HER2 (1+: 35.5 %; 2+: 18.2 %; 3+: 5.2 %). Validity of the results was confirmed using the HercepTest antibody. Higher HER2 expression was associated with a higher Gleason Score/Grade Group and advanced disease. CONCLUSIONS: Our findings support the adverse prognostic impact on HER2 in PCa. We propose the use of a modified scoring system to evaluate HER2 expression in PCa. The observed high prevalence of HER2 expression supports the consideration of novel HER2-targeted therapies addressing even low levels of HER2 expression in future PCa trials.

Localized DNA demethylation at recombination intermediates during immunoglobulin heavy chain gene assembly.

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D(H)) and joining (J(H)) gene segments were methylated, DJ(H) junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eµ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination.

RAGs' eye view of the immunoglobulin heavy chain gene locus.

The immunoglobulin heavy chain (IgH) gene locus is activated at a precise stage of B lymphocyte development to undergo gene rearrangements that assemble the functional gene. In this review we summarize our current understanding of the chromatin state of the IgH as it appears just prior to the initiation of V(D)J recombination, and the implications of this structure for regulation of recombination. We also examine the role of the intron enhancer, Eµ, in establishing the pre-rearrangement chromatin structure. The emerging picture shows that the IgH locus consists of independently regulated domains, each of which requires multiple levels of epigenetic changes to reach the fully activated state.

Disparities in Acute Lymphocytic Leukemia Outcomes Among Young Adults.

Background: Age is a strong prognostic factor in acute lymphocytic leukemia (ALL), with children doing better than adults with the same disease. One hypothesis for this age-based disparity is differences in treatment regimens. Optimizing care for adolescents and young adults (AYA) with ALL has not been well defined and disparities in care exist. We conducted a retrospective study of all veterans with ALL diagnosed between the ages of 18 and 45 since the year 2000 to evaluate disparities among prognostication methods, treatment regimens, and accrual to clinical trials with regard to age and race/ethnicity and how these factors influence overall survival. Methods: Electronic medical record data from the VA Informatics and Computing Infrastructure (VINCI) were used to identify 6,724 patients with an ICD-9 or 10 code for ALL. All patients were chart checked to confirm an ALL diagnosis between the ages of 18 and 45 and excluded if they were diagnosed before 2000, had childhood ALL, or if induction protocol was not recorded. A total of 252 patients were included in the final analysis. Multivariate analysis was performed with controls for age, ALL subtype (B, T, mixed phenotype), Ph status, cytogenetic risk (based on modified Medical Research Council-Eastern Cooperative Oncology Group (MRC-ECOG) study), obesity (body mass index (BMI) > 30), and race. Results: Patients treated with pediatric regimens, including pediatric-inspired regimens, have statistically significant (P = 0.009) survival gains, with a hazard ratio (HR) of 0.52 after controlling for age, obesity, ALL subtype, cytogenetic risk and race. White patients had significantly improved OS compared to people of color (HR 0.57, P = 0.02) after controlling for the aforementioned covariates. Black patients were far less likely (23%) to receive a transplant than non-Black patients (46%). Only 7% of patients were treated on a clinical trial. Conclusions: These data demonstrate that treatment with a pediatric regimen significantly improves overall survival in patients up to the age of 45 and suggests ongoing shortcomings in treatment for young adults with ALL, especially 30 to 45 years old, including persistently high use of adult induction regimens, low rates of referral to clinical trials, and significant racial disparities in bone marrow transplants for Black patients.

Epigenetic features that regulate IgH locus recombination and expression.

Precisely regulated rearrangements that yield imprecise recombination junctions are hallmarks of antigen receptor gene assembly. At the immunoglobulin heavy chain (IgH) gene locus this is initiated by rearrangement of a D (H) gene segment to a J (H) gene segment to generate DJ(H) junctions, followed by rearrangement of a V (H) gene segment to the DJ(H) junction to generate fully recombined VDJ alleles. In this review we discuss the regulatory features of each step of IgH gene assembly and the role of epigenetic mechanisms in achieving regulatory precision.

Cutting edge: SWI/SNF mediates antisense Igh transcription and locus-wide accessibility in B cell precursors.

The stepwise process of Ag receptor gene assembly, termed V(D)J recombination, is coordinated during lymphocyte development by sweeping changes in chromatin that permit or deny access to a single recombinase enzyme. We now show that switching/sucrose nonfermenting (SWI/SNF) chromatin remodeling complexes are recruited to the Igh locus by an enhancer-dependent process and that these complexes are essential for generating recombinase accessibility throughout the locus. Depletion of SWI/SNF in pro-B cells also inhibits antisense transcription through all clusters of Igh gene segments, a pioneering process that has been implicated in the initial opening of chromatin. We conclude that SWI/SNF complexes play multiple roles in Igh gene assembly, ranging from initial locus activation to the spreading and maintenance of chromatin accessibility over large V(H), D(H), and J(H) domains.

Why African Americans say "No": A Study of Pharmacogenomic Research Participation.

Objective: To identify reasons for nonparticipation by African Americans in cardiovascular pharmacogenomic research. Design: Prospective, open-ended, qualitative survey. Setting: Research staff approached patients eligible for the Discovery Project of The African American Cardiovascular pharmacogenomics CONsorTium in the inpatient or outpatient setting at four different institutions during September and October 2018. Participants: Potential Discovery Project participants self-identified as African American, aged >18 years, were on one of five cardiovascular drugs of interest, and declined enrollment in the Discovery Project. Main Outcome Measures: Reasons for nonparticipation. Methods: After declining participation in the Discovery Project, patients were asked, "What are your reasons for not participating?" We analyzed their responses using a directed content analytic approach. Ultimately, responses were coded into one of nine categories and analyzed using descriptive statistics. Results: Of the 194 people approached for the Discovery Project during an eight-week period, 82 declined participation and provided information for this study. The most common reason for refusal was concern about the amount of blood drawn (19.5%). The next most common reasons for refusal to participate included concerns about genetic testing (14.6%) and mistrust of research (12.2%). Across study sites, significantly more patients enrolled in the inpatient than outpatient setting (P<.001). Significantly more women and younger individuals declined participation due to concerns about genetic testing and too little compensation (P<.05). Conclusions: Collection of blood samples and concerns about genetic testing are obstacles for the recruitment of African Americans to pharmacogenomics studies. Efforts to overcome these barriers to participation are needed to improve representation of minorities in pharmacogenomic research. Enrolling participants from inpatient populations may be a solution to bolster recruitment efforts.

Oncogenic NRAS, KRAS, and HRAS exhibit different leukemogenic potentials in mice.

RAS proteins are small GTPases that play a central role in transducing signals that regulate cell proliferation, survival, and differentiation. The RAS proteins interact with a common set of activators and effectors; however, they associate with different microdomains of the plasma membrane as well as other endomembranes and are capable of generating distinct signal outputs. Mutations that result in constitutive activation of RAS proteins are associated with approximately 30% of all human cancers; however, different RAS oncogenes are preferentially associated with different types of human cancer. In myeloid malignancies, NRAS mutations are more frequent than KRAS mutations, whereas HRAS mutations are rare. The mechanism underlying the different frequencies of RAS isoforms mutated in myeloid leukemia is not known. In this study, we compared the leukemogenic potential of activated NRAS, KRAS, and HRAS in the same bone marrow transduction/transplantation model system. We found that all three RAS oncogenes have the ability to induce myeloid leukemias, yet have distinct leukemogenic strengths and phenotypes. The models established here provide a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing targeted therapies.

A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation.

A tissue-specific transcriptional enhancer, Emu, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Emu results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Emu-independent and Emu-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression.

Beta-catenin expression results in p53-independent DNA damage and oncogene-induced senescence in prelymphomagenic thymocytes in vivo.

The expression of beta-catenin, a potent oncogene, is causally linked to tumorigenesis. Therefore, it was surprising that the transgenic expression of oncogenic beta-catenin in thymocytes resulted in thymic involution instead of lymphomagenesis. In this report, we demonstrate that this is because the expression of oncogenic beta-catenin induces DNA damage, growth arrest, oncogene-induced senescence (OIS), and apoptosis of immature thymocytes. In p53-deficient mice, the expression of oncogenic beta-catenin still results in DNA damage and OIS, but the thymocytes survive and eventually progress to thymic lymphoma. beta-Catenin-induced thymic lymphomas are distinct from lymphomas that arise in p53(-/-) mice. They are CD4(-) CD8(-), while p53-dependent lymphomas are largely CD4(+) CD8(+), and they develop at an earlier age and in the absence of c-Myc expression or Notch1 signaling. Thus, we report that oncogenic beta-catenin-induced, p53-independent growth arrest and OIS and p53-dependent apoptosis protect developing thymocytes from transformation by oncogenic beta-catenin.

p16(INK4a) translation suppressed by miR-24.

BACKGROUND: Expression of the tumor suppressor p16(INK4a) increases during aging and replicative senescence. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that the microRNA miR-24 suppresses p16 expression in human diploid fibroblasts and cervical carcinoma cells. Increased p16 expression with replicative senescence was associated with decreased levels of miR-24, a microRNA that was predicted to associate with the p16 mRNA coding and 3'-untranslated regions. Ectopic miR-24 overexpression reduced p16 protein but not p16 mRNA levels. Conversely, introduction of antisense (AS)-miR-24 blocked miR-24 expression and markedly enhanced p16 protein levels, p16 translation, and the production of EGFP-p16 reporter bearing the miR-24 target recognition sites. CONCLUSIONS/SIGNIFICANCE: Together, our results suggest that miR-24 represses the initiation and elongation phases of p16 translation.

A plant homeodomain in RAG-2 that binds Hypermethylated lysine 4 of histone H3 is necessary for efficient antigen-receptor-gene rearrangement.

V(D)J recombination is initiated by the recombination activating gene (RAG) proteins RAG-1 and RAG-2. The ability of antigen-receptor-gene segments to undergo V(D)J recombination is correlated with spatially- and temporally-restricted chromatin modifications. We have found that RAG-2 bound specifically to histone H3 and that this binding was absolutely dependent on dimethylation or trimethylation at lysine 4 (H3K4me2 or H3K4me3). The interaction required a noncanonical plant homeodomain (PHD) that had previously been described within the noncore region of RAG-2. Binding of the RAG-2 PHD finger to chromatin across the IgH D-J(H)-C locus showed a strong correlation with the distribution of trimethylated histone H3 K4. Mutation of a conserved tryptophan residue in the RAG-2 PHD finger abolished binding to H3K4me3 and greatly impaired recombination of extrachromosomal and endogenous immunoglobulin gene segments. Together, these findings are consistent with the interpretation that recognition of hypermethylated histone H3 K4 promotes efficient V(D)J recombination in vivo.

Repeat organization and epigenetic regulation of the DH-Cmu domain of the immunoglobulin heavy-chain gene locus.

The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.

Oncogenic NRAS rapidly and efficiently induces CMML- and AML-like diseases in mice.

Activating mutations in RAS, predominantly NRAS, are common in myeloid malignancies. Previous studies in animal models have shown that oncogenic NRAS is unable to induce myeloid malignancies effectively, and it was suggested that oncogenic NRAS might only act as a secondary mutation in leukemogenesis. In this study, we examined the leukemogenicity of NRAS using an improved mouse bone marrow transduction and transplantation model. We found that oncogenic NRAS rapidly and efficiently induced chronic myelomonocytic leukemia (CMML)- or acute myeloid leukemia (AML)- like disease in mice, indicating that mutated NRAS can function as an initiating oncogene in the induction of myeloid malignancies. In addition to CMML and AML, we found that NRAS induced mastocytosis in mice. This result indicates that activation of the RAS pathway also plays an important role in the pathogenesis of mastocytosis. The mouse model for NRAS leukemogenesis established here provides a system for further studying the molecular mechanisms in the pathogenesis of myeloid malignancies and for testing relevant therapies.