MYC-rearranged mature B-cell lymphomas in children and young adults are molecularly Burkitt Lymphoma.

Aggressive B-cell non-Hodgkin lymphomas (NHL) in children, adolescents, and young adults (CAYA) include Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), and a subset of high-grade tumors with features intermediate between these entities whose genetic and molecular profiles have not been completely elucidated. In this study, we have characterized 37 aggressive B-NHL in CAYA, 33 with high-grade morphology, and 4 DLBCL with MYC rearrangement (MYC-R), using targeted next-generation sequencing and the aggressive lymphoma gene expression germinal center B-cell-like (GCB), activated B-cell-like (ABC), and dark zone signatures (DZsig). Twenty-two tumors had MYC-R without BCL2 breaks, and two MYC-non-R cases had BCL6 translocations. MYC-R cases, including DLBCL, carried BL-related mutations and copy number alterations. Conversely, MYC-non-R lymphomas had alterations in the B-cell receptor signaling/NF-κB pathway (71%). DZsig was expressed in 12/13 of MYC-R tumors but only in 2/10 of MYC-non-R GCB tumors (P < 0.001). The 3-year event-free survival (EFS) of the whole cohort was 79.6%. TP53 and KMT2C mutations conferred inferior outcome (3-year EFS P < 0.05). Overall, MYC-R lymphomas in CAYA have a molecular profile similar to BL regardless of their high-grade or DLBCL morphology, whereas MYC-non-R has more heterogeneous genetic alterations closer to that of DLBCL.

The DNA loop release factor WAPL suppresses Epstein-Barr virus latent membrane protein expression to maintain the highly restricted latency I program.

Epstein-Barr virus (EBV) uses latency programs to colonize the memory B-cell reservoir, and each program is associated with human malignancies. However, knowledge remains incomplete of epigenetic mechanisms that maintain the highly restricted latency I program, present in memory and Burkitt lymphoma cells, in which EBNA1 is the only EBV-encoded protein expressed. Given increasing appreciation that higher order chromatin architecture is an important determinant of viral and host gene expression, we investigated roles of Wings Apart-Like Protein Homolog (WAPL), a host factor that unloads cohesin to control DNA loop size and that was discovered as an EBNA2-associated protein. WAPL knockout (KO) in Burkitt cells de-repressed LMP1 and LMP2A expression, but not other EBV oncogenes, to yield a viral program reminiscent of EBV latency II, which is rarely observed in B-cells. WAPL KO also increased LMP1/2A levels in latency III lymphoblastoid cells. WAPL KO altered EBV genome architecture, triggering formation of DNA loops between the LMP promoter region and the EBV origins of lytic replication (oriLyt). Hi-C analysis further demonstrated that WAPL KO reprogrammed EBV genomic DNA looping. LMP1 and LMP2A de-repression correlated with decreased histone repressive marks at their promoters. We propose that EBV coopts WAPL to negatively regulate latent membrane protein expression to maintain Burkitt latency I.

Obesity increases genomic instability at DNA repeat-mediated endogenous mutation hotspots.

Obesity is associated with increased cancer risk, yet the underlying mechanisms remain elusive. Obesity-associated cancers involve disruptions in metabolic and cellular pathways, which can lead to genomic instability. Repetitive DNA sequences capable of adopting alternative DNA structures (e.g., H-DNA) stimulate mutations and are enriched at mutation hotspots in human cancer genomes. However, it is not known if obesity impacts DNA repeat-mediated endogenous mutation hotspots. We address this gap by measuring mutation frequencies in obese and normal-weight transgenic reporter mice carrying either a control human B-DNA- or an H-DNA-forming sequence (from a translocation hotspot in c-MYC in Burkitt lymphoma). Here, we discover that H-DNA-induced DNA damage and mutations are elevated in a tissue-specific manner, and DNA repair efficiency is reduced in obese mice compared to those on the control diet. These findings elucidate the impact of obesity on cancer-associated endogenous mutation hotspots, providing mechanistic insight into the link between obesity and cancer.

Targeted eradication of EBV-positive cancer cells by CRISPR/dCas9-mediated EBV reactivation in combination with ganciclovir.

Epstein-Barr virus (EBV) is a ubiquitous human tumor virus that establishes lifelong, persistent infections in B cells. The presence of EBV in cancer cells presents an opportunity to target these cells by reactivating the virus from latency. In this study, we developed a novel approach for EBV reactivation termed clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated EBV reactivation (CMER) strategy. Using modified CRISPR-associated protein 9 (dCas9) fused with VP64, we designed 10 single guide RNAs (sgRNAs) to target and activate the EBV immediate-early gene promoter. In Akata Burkitt lymphoma cells, 9 out of 10 CMER sgRNAs effectively reactivated EBV. Among these, CMER sgRNA-5 triggered robust reactivation across various cell types, including lymphoma, gastric cancer, and nasopharyngeal carcinoma cells. Importantly, the combination of CMER and ganciclovir selectively eliminated EBV-positive cells, regardless of their cell origin. These findings indicate that targeted virus reactivation by CMER, combined with nucleoside analog therapy, holds promise for EBV-associated cancer treatment. IMPORTANCE: This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Barr virus in cancer cells. EBV is associated with various cancers, and reactivating EBV from latency offers a potential therapeutic strategy. We utilized an enzymatically inactive CRISPR-associated protein 9 (dCas9) fused with VP64 and designed 10 single guide RNAs to target the EBV immediate-early gene promoter. Nine of these sgRNAs effectively reactivated EBV in Burkitt lymphoma cells, with CMER sgRNA-5 demonstrating strong reactivation across different cancer cell types. Combining CMER with ganciclovir selectively eliminated EBV-positive cells, showing promise for EBV-associated cancer treatment.

Tumor microenvironment of Burkitt lymphoma: different immune signatures with different clinical behavior.

ABSTRACT: Burkitt lymphoma (BL) is characterized by a tumor microenvironment (TME) in which macrophages represent the main component, determining a distinct histological appearance known as "starry sky" pattern. However, in some instances, BL may exhibit a granulomatous reaction that has been previously linked to favorable prognosis and spontaneous regression. The aim of our study was to deeply characterize the immune landscape of 7 cases of Epstein-Barr virus-positive (EBV+) BL with granulomatous reaction compared with 8 cases of EBV+ BL and 8 EBV-negative (EBV-) BL, both with typical starry sky pattern, by Gene expression profiling performed on the NanoString nCounter platform. Subsequently, the data were validated using multiplex and combined immunostaining. Based on unsupervised clustering of differentially expressed genes, BL samples formed 3 distinct clusters differentially enriched in BL with a diffuse granulomatous reaction (cluster 1), EBV+ BL with typical starry sky pattern (cluster 2), EBV- BL with typical "starry sky" (cluster 3). We observed variations in the immune response signature among BL with granulomatous reaction and BL with typical "starry sky," both EBV+ and EBV-. The TME signature in BL with diffuse granulomatous reaction showed a proinflammatory response, whereas BLs with "starry sky" were characterized by upregulation of M2 polarization and protumor response. Moreover, the analysis of additional signatures revealed an upregulation of the dark zone signature and epigenetic signature in BL with a typical starry sky. Tumor-associated macrophages and epigenetic regulators may be promising targets for additional therapies for BL lymphoma, opening novel immunotherapeutic strategies.

MicroRNA-focused CRISPR/Cas9 screen identifies miR-142 as a key regulator of Epstein-Barr virus reactivation.

Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle.

The F-box E3 ligase protein FBXO11 regulates EBNA3C-associated degradation of BCL6.

Most mature B-cell malignancies originate from the malignant transformation of germinal center (GC) B cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is BCL6, a key regulator of this process. We now demonstrate that BCL6 protein levels were dramatically decreased in Epstein-Barr virus (EBV)-positive lymphoblastoid cell lines and Burkitt's lymphoma cell lines. Notably, BCL6 degradation was significantly enhanced in the presence of both EBNA3C and FBXO11. Furthermore, the amino-terminal domain of EBNA3C, which contains residues 50-100, interacts directly with FBXO11. The expression of EBNA3C and FBXO11 resulted in a significant induction of cell proliferation. Furthermore, BCL6 protein expression levels were regulated by EBNA3C via the Skp Cullin Fbox (SCF)FBXO11 complex, which mediated its ubiquitylation, and knockdown of FBXO11 suppressed the transformation of lymphoblastoid cell lines. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction and raise the possibility of developing new targeted therapies against EBV-associated cancers. IMPORTANCE: The novel revelation in our study involves the suppression of BCL6 expression by the essential Epstein-Barr virus (EBV) antigen EBNA3C, shedding new light on our current comprehension of how EBV contributes to lymphomagenesis by impeding the germinal center reaction. It is crucial to note that while several EBV latent proteins are expressed in infected cells, the collaborative mechanisms among these proteins in regulating B-cell development or inducing B-cell lymphoma require additional investigation. Nonetheless, our findings carry significance for the development of emerging strategies aimed at addressing EBV-associated cancers.

Molecular targets of glucocorticoids that elucidate their therapeutic efficacy in aggressive lymphomas.

Glucocorticoids have been used for decades to treat lymphomas without an established mechanism of action. Using functional genomic, proteomic, and chemical screens, we discover that glucocorticoids inhibit oncogenic signaling by the B cell receptor (BCR), a recurrent feature of aggressive B cell malignancies, including diffuse large B cell lymphoma and Burkitt lymphoma. Glucocorticoids induce the glucocorticoid receptor (GR) to directly transactivate genes encoding negative regulators of BCR stability (LAPTM5; KLHL14) and the PI3 kinase pathway (INPP5D; DDIT4). GR directly represses transcription of CSK, a kinase that limits the activity of BCR-proximal Src-family kinases. CSK inhibition attenuates the constitutive BCR signaling of lymphomas by hyperactivating Src-family kinases, triggering their ubiquitination and degradation. With the knowledge that glucocorticoids disable oncogenic BCR signaling, they can now be deployed rationally to treat BCR-dependent aggressive lymphomas and used to construct mechanistically sound combination regimens with inhibitors of BTK, PI3 kinase, BCL2, and CSK.

Transcriptome analysis of Burkitt lymphoma cells treated with anti-convulsant drugs that are inhibitors of Epstein-Barr virus lytic reactivation.

Herpesviruses have two distinct life cycle stages, latency and lytic replication. Epstein-Barr virus (EBV), a gamma-herpesvirus, establishes latency in vivo and in cultured cells. Cell lines harboring latent EBV can be induced into the lytic cycle by treatment with chemical inducing agents. In the Burkitt lymphoma cell line HH514-16 the viral lytic cycle is triggered by butyrate, a histone deacetylase (HDAC) inhibitor. Butyrate also alters expression of thousands of cellular genes. However, valproic acid (VPA), another HDAC inhibitor with global effects on cellular gene expression blocks EBV lytic gene expression in Burkitt lymphoma cell lines. Valpromide (VPM), an amide derivative of VPA, is not an HDAC inhibitor, but like VPA blocks induction of the EBV lytic cycle. VPA and VPM are the first examples of inhibitors of initial stages of lytic reactivation. We compared the effects of VPA and VPM, alone and in combination with butyrate, on host cellular gene expression using whole transcriptome analysis (RNA-seq). Gene expression was analyzed 6 h after addition of the compounds, a time before the first EBV lytic transcripts are detected. The results address two alternative, yet possibly complementary, mechanisms for regulation of EBV lytic reactivation. First, cellular genes that were up- or down-regulated by butyrate, but no longer altered in the presence of VPA or VPM, represent genes that correlated with EBV lytic reactivation. Second, genes regulated similarly by VPA and VPM in the absence and presence of butyrate are candidates for suppressors of EBV reactivation. Two genes upregulated by the lytic cycle inhibitors, CHAC1 and SLC7A11, are related to redox status and the iron-dependent cell death pathway ferroptosis. This study generates new hypotheses for control of the latency to lytic cycle switch of EBV and provides the first description of effects of the anti-convulsant drug VPM on global human cellular gene expression.

SOX11 expression is restricted to EBV-negative Burkitt lymphoma and is associated with molecular genetic features.

ABSTRACT: SRY-related HMG-box gene 11 (SOX11) is a transcription factor overexpressed in mantle cell lymphoma (MCL), a subset of Burkitt lymphomas (BL) and precursor lymphoid cell neoplasms but is absent in normal B cells and other B-cell lymphomas. SOX11 has an oncogenic role in MCL but its contribution to BL pathogenesis remains uncertain. Here, we observed that the presence of Epstein-Barr virus (EBV) and SOX11 expression were mutually exclusive in BL. SOX11 expression in EBV-negative (EVB-) BL was associated with an IG∷MYC translocation generated by aberrant class switch recombination, whereas in EBV-negative (EBV-)/SOX11-negative (SOX11-) tumors the IG∷MYC translocation was mediated by mistaken somatic hypermutations. Interestingly, EBV- SOX11-expressing BL showed higher frequency of SMARCA4 and ID3 mutations than EBV-/SOX11- cases. By RNA sequencing, we identified a SOX11-associated gene expression profile, with functional annotations showing partial overlap with the SOX11 transcriptional program of MCL. Contrary to MCL, no differences on cell migration or B-cell receptor signaling were found between SOX11- and SOX11-positive (SOX11+) BL cells. However, SOX11+ BL showed higher adhesion to vascular cell adhesion molecule 1 (VCAM-1) than SOX11- BL cell lines. Here, we demonstrate that EBV- BL comprises 2 subsets of cases based on SOX11 expression. The mutual exclusion of SOX11 and EBV, and the association of SOX11 with a specific genetic landscape suggest a role of SOX11 in the early pathogenesis of BL.

Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies.

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and natural killer (NK) cells have shown remarkable clinical efficacy in treating hematological malignancies; however, current methods mainly utilize viral vectors that are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure-guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof-of-principle TcB-M engineering of CAR-NK and CAR-T cells shows low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Latent Epstein-Barr virus infection collaborates with Myc over-expression in normal human B cells to induce Burkitt-like Lymphomas in mice.

Epstein-Barr virus (EBV) is an important cause of human lymphomas, including Burkitt lymphoma (BL). EBV+ BLs are driven by Myc translocation and have stringent forms of viral latency that do not express either of the two major EBV oncoproteins, EBNA2 (which mimics Notch signaling) and LMP1 (which activates NF-κB signaling). Suppression of Myc-induced apoptosis, often through mutation of the TP53 (p53) gene or inhibition of pro-apoptotic BCL2L11 (BIM) gene expression, is required for development of Myc-driven BLs. EBV+ BLs contain fewer cellular mutations in apoptotic pathways compared to EBV-negative BLs, suggesting that latent EBV infection inhibits Myc-induced apoptosis. Here we use an EBNA2-deleted EBV virus (ΔEBNA2 EBV) to create the first in vivo model for EBV+ BL-like lymphomas derived from primary human B cells. We show that cord blood B cells infected with both ΔEBNA2 EBV and a Myc-expressing vector proliferate indefinitely on a CD40L/IL21 expressing feeder layer in vitro and cause rapid onset EBV+ BL-like tumors in NSG mice. These LMP1/EBNA2-negative Myc-driven lymphomas have wild type p53 and very low BIM, and express numerous germinal center B cell proteins (including TCF3, BACH2, Myb, CD10, CCDN3, and GCSAM) in the absence of BCL6 expression. Myc-induced activation of Myb mediates expression of many of these BL-associated proteins. We demonstrate that Myc blocks LMP1 expression both by inhibiting expression of cellular factors (STAT3 and Src) that activate LMP1 transcription and by increasing expression of proteins (DNMT3B and UHRF1) known to enhance DNA methylation of the LMP1 promoters in human BLs. These results show that latent EBV infection collaborates with Myc over-expression to induce BL-like human B-cell lymphomas in mice. As NF-κB signaling retards the growth of EBV-negative BLs, Myc-mediated repression of LMP1 may be essential for latent EBV infection and Myc translocation to collaboratively induce human BLs.

Targeted therapy in Burkitt lymphoma: Small molecule inhibitors under investigation.

Multiagent chemoimmunotherapy remains the standard of care treatment for Burkitt lymphoma leading to a cure in the majority of cases. However, frontline treatment regimens are associated with a significant risk of treatment related toxicity especially in elderly and immunocompromised patients. Additionally, prognosis remains dismal in refractory/relapsed Burkitt lymphoma. Thus, novel therapies are required to not only improve outcomes in relapsed/refractory Burkitt lymphoma but also minimize frontline treatment related toxicities. Recurrent genomic changes and signalling pathway alterations that have been implicated in the Burkitt lymphomagenesis include cell cycle dysregulation, cell proliferation, inhibition of apoptosis, epigenetic dysregulation and tonic B-cell receptor-phosphatidylinositol 3-kinase (BCR-PI3K) signalling. Here, we will discuss novel targeted therapy approaches using small molecule inhibitors that could pave the way to the future treatment landscape based on the understanding of recurrent genomic changes and signalling pathway alterations in the lymphomagenesis of adult Burkitt lymphoma.

Cytogenetic and pathologic characterization of MYC-rearranged B-cell lymphomas in pediatric and young adult patients.

MYC-rearranged B-cell lymphoma (BCL) in the pediatric/young adult (YA) age group differs substantially in disease composition from adult cohorts. However, data regarding the partner genes, concurrent rearrangements, and ultimate diagnoses in these patients is scarce compared to that in adult cohorts. We aimed to characterize the spectrum of MYC-rearranged (MYC-R) mature, aggressive BCL in the pediatric/YA population. A retrospective study of morphologic, immunophenotypic, and fluorescence in situ hybridization (FISH) results of patients age ≤ 30 years with suspected Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) or high-grade B-cell lymphoma (HGBCL), and a MYC-R by FISH between 2013-2022 was performed. Two-hundred fifty-eight cases (129 (50%) pediatric (< 18 years) and 129 (50%) YA (18-30 years)) were included. Most MYC-R BCL in pediatric (89%) and YA (66%) cases were BL. While double-hit (DH) cytogenetics (MYC with BCL2 and/or BCL6-R, HGBCL-DH) was rare in the pediatric population (2/129, 2%), HGBCL-DH increased with age and was identified in 17/129 (13%) of YA cases. Most HGBCL-DH had MYC and BCL6-R, while BCL2-R were rare in both groups (3/258, 1%). MYC-R without an IG partner was more common in the YA group (14/116 (12%) vs 2/128 (2%), p = 0.001). The pediatric to YA transition is characterized by decreasing frequency in BL and increasing genetic heterogeneity of MYC-R BCL, with emergence of DH-BCL with MYC and BCL6-R. FISH to evaluate for BCL2 and BCL6 rearrangements is likely not warranted in the pediatric population but should continue to be applied in YA BCL.

Childhood and Adolescent Relapsed/Refractory Aggressive B-Cell Lymphomas With t(8;14) and BCL2 Expression, Burkitt Lymphoma Versus Diffuse Large B-Cell Lymphoma: A Diagnostic Challenge.

We present 2 diagnostically challenging cases of pediatric/adolescent relapsed/refractory aggressive mature B-cell non-Hodgkin lymphoma (B-NHL) within the spectrum of Burkitt lymphoma and diffuse large B-cell lymphoma and illustrate the different therapeutic regimens that are employed for pediatric and adult cancer centers. Both cases displayed varying-sized lymphoma cells with occasional single prominent nucleoli and heterogeneous BCL2 expression. Cytogenetics revealed complex karyotypes with t(8:14)(q24.2;q32) and IGH::MYC rearrangement by FISH. Next generation sequencing revealed deleterious TP53 and MYC mutations. We concluded that both could be diagnosed as "DLBCL-NOS with MYC rearrangement" using the current pathologic classifications, 2022 International Consensus Classification (ICC) and World Health Organization Classifications of Haematolymphoid Tumors (WHO-HAEM5). This report illustrates diagnostic challenges and treatment dilemmas that may be encountered, particularly for adolescent and young adults (AYA).