Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCCs) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The former group exhibits reduced proliferation, genome instability, and heightened sensitivity to genotoxic agents like PARP1/2 inhibitors. Conversely, H3K36M HNSCC models with constant H3K27me3 levels lack these characteristics unless H3K27me3 is elevated by DNA hypomethylating agents or inhibiting H3K27me3 demethylases KDM6A/B. Mechanistically, H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, aberrant H3K27me3 levels induced by H3K36M expression are not a bona fide epigenetic mark because they require continuous expression of H3K36M to be inherited. Moreover, increased sensitivity to PARP1/2 inhibitors in H3K36M HNSCC models depends solely on elevated H3K27me3 levels and diminishing BRCA1- and FANCD2-dependent DNA repair. Finally, a PARP1/2 inhibitor alone reduces tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a model with consistent H3K27me3, a combination of PARP1/2 inhibitors and agents that up-regulate H3K27me3 proves to be successful. These findings underscore the crucial balance between H3K36 and H3K27 methylation in maintaining genome instability, offering new therapeutic options for patients with H3K36me-deficient tumors.

PRAME induces genomic instability in uveal melanoma.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME that can be targeted therapeutically in cancer.

Methylation of histone H3 lysine 36 is a barrier for therapeutic interventions of head and neck squamous cell carcinoma.

Approximately 20% of head and neck squamous cell carcinomas (HNSCC) exhibit reduced methylation on lysine 36 of histone H3 (H3K36me) due to mutations in histone methylase NSD1 or a lysine-to-methionine mutation in histone H3 (H3K36M). Whether such alterations of H3K36me can be exploited for therapeutic interventions is still unknown. Here, we show that HNSCC models expressing H3K36M can be divided into two groups: those that display aberrant accumulation of H3K27me3 and those that maintain steady levels of H3K27me3. The first group shows decreased proliferation, genome instability, and increased sensitivity to genotoxic agents, such as PARP1/2 inhibitors. In contrast, the H3K36M HNSCC models with steady H3K27me3 levels do not exhibit these characteristics unless H3K27me3 levels are elevated, either by DNA hypomethylating agents or by inhibiting the H3K27me3 demethylases KDM6A/B. Mechanistically, we found that H3K36M reduces H3K36me by directly impeding the activities of the histone methyltransferase NSD3 and the histone demethylase LSD2. Notably, we found that aberrant H3K27me3 levels induced by H3K36M expression is not a bona fide epigenetic mark in HNSCC since it requires continuous expression of H3K36M to be inherited. Moreover, increased sensitivity of H3K36M HNSCC models to PARP1/2 inhibitors solely depends on the increased H3K27me3 levels. Indeed, aberrantly high H3K27me3 levels decrease BRCA1 and FANCD2-dependent DNA repair, resulting in higher sensitivity to DNA breaks and replication stress. Finally, in support of our in vitro findings, a PARP1/2 inhibitor alone reduce tumor burden in a H3K36M HNSCC xenograft model with elevated H3K27me3, whereas in a H3K36M HNSCC xenograft model with consistent H3K27me3 levels, a combination of PARP1/2 inhibitors and agents that upregulate H3K27me3 proves to be successful. In conclusion, our findings underscore a delicate balance between H3K36 and H3K27 methylation, essential for maintaining genome stability. This equilibrium presents promising therapeutic opportunities for patients with H3K36me-deficient tumors.

PRAME induces genomic instability in uveal melanoma.

PRAME is a CUL2 ubiquitin ligase subunit that is normally expressed in the testis but becomes aberrantly overexpressed in many cancer types in association with aneuploidy and metastasis. Here, we show that PRAME is expressed predominantly in spermatogonia around the time of meiotic crossing-over in coordination with genes mediating DNA double strand break repair. Expression of PRAME in somatic cells upregulates pathways involved in meiosis, chromosome segregation and DNA repair, and it leads to increased DNA double strand breaks, telomere dysfunction and aneuploidy in neoplastic and non-neoplastic cells. This effect is mediated at least in part by ubiquitination of SMC1A and altered cohesin function. PRAME expression renders cells susceptible to inhibition of PARP1/2, suggesting increased dependence on alternative base excision repair pathways. These findings reveal a distinct oncogenic function of PRAME than can be targeted therapeutically in cancer.

Oxidized mC modulates synthetic lethality to PARP inhibitors for the treatment of leukemia.

TET2 haploinsufficiency is a driving event in myeloid cancers and is associated with a worse prognosis in patients with acute myeloid leukemia (AML). Enhancing residual TET2 activity using vitamin C increases oxidized 5-methylcytosine (mC) formation and promotes active DNA demethylation via base excision repair (BER), which slows leukemia progression. We utilize genetic and compound library screening approaches to identify rational combination treatment strategies to improve use of vitamin C as an adjuvant therapy for AML. In addition to increasing the efficacy of several US Food and Drug Administration (FDA)-approved drugs, vitamin C treatment with poly-ADP-ribosyl polymerase inhibitors (PARPis) elicits a strong synergistic effect to block AML self-renewal in murine and human AML models. Vitamin-C-mediated TET activation combined with PARPis causes enrichment of chromatin-bound PARP1 at oxidized mCs and γH2AX accumulation during mid-S phase, leading to cell cycle stalling and differentiation. Given that most AML subtypes maintain residual TET2 expression, vitamin C could elicit broad efficacy as a PARPi therapeutic adjuvant.

Endocrine resistance and breast cancer plasticity are controlled by CoREST.

Resistance to cancer treatment remains a major clinical hurdle. Here, we demonstrate that the CoREST complex is a key determinant of endocrine resistance and ER+ breast cancer plasticity. In endocrine-sensitive cells, CoREST is recruited to regulatory regions co-bound to ERα and FOXA1 to regulate the estrogen pathway. In contrast, during temporal reprogramming towards a resistant state, CoREST is recruited to AP-1 sites. In reprogrammed cells, CoREST favors chromatin opening, cJUN binding to chromatin, and gene activation by controlling SWI/SNF recruitment independently of the demethylase activity of the CoREST subunit LSD1. Genetic and pharmacological CoREST inhibition reduces tumorigenesis and metastasis of endocrine-sensitive and endocrine-resistant xenograft models. Consistently, CoREST controls a gene signature involved in invasiveness in clinical breast tumors resistant to endocrine therapies. Our studies reveal CoREST functions that are co-opted to drive cellular plasticity and resistance to endocrine therapies and tumorigenesis, thus establishing CoREST as a potential therapeutic target for the treatment of advanced breast cancer.

LMO2 expression is frequent in T-lymphoblastic leukemia and correlates with survival, regardless of T-cell stage.

T- lymphoblastic leukemia/lymphoma (T-LL) is an aggressive malignancy of immature T-cells with poor overall survival (OS) and in need of new therapies. LIM-domain only 2 (LMO2) is a critical regulator of hematopoietic cell development that can be overexpressed in T-LL due to chromosomal abnormalities. Deregulated LMO2 expression contributes to T-LL development by inducing block of T-cell differentiation and continuous thymocyte self-renewal. However, LMO2 expression and its biologic significance in T-LL remain largely unknown. We analyzed LMO2 expression in 100 initial and follow-up biopsies of T-LL from 67 patients, including 31 (46%) early precursor T-cell (ETP)-ALL, 26 (39%) cortical and 10 (15%) medullary type. LMO2 expression was present in 50 (74.6%) initial biopsies with an average of 87% positive tumor cells (range 30-100%). LMO2 expression in ETP, medullary and cortical T-LLs was not statistically different. In patients with biopsies after initial therapy, LMO2 expression was stable. LMO2 expression was associated with longer OS (p = 0.048) regardless of T-lymphoblast stage or other clinicopathologic features. These findings indicate that LMO2 is a promising new prognostic marker that could predict patients' outcomes and potentially be targeted for novel chemotherapy, i.e. PARP1/2 inhibitors, which have been shown to enhance chemotherapy sensitivity in LMO2 expressing diffuse large B cell lymphoma (DLBCL) tumors by decreasing DNA repair efficiency.

Kaposi's sarcoma herpesvirus activates the hypoxia response to usurp HIF2α-dependent translation initiation for replication and oncogenesis.

Kaposi's sarcoma herpesvirus (KSHV) is an angiogenesis-inducing oncovirus whose ability to usurp the oxygen-sensing machinery is central to its oncogenicity. By upregulating the hypoxia-inducible factors (HIFs), KSHV reprograms infected cells to a hypoxia-like state, triggering angiogenesis. Here we identify a link between KSHV replicative biology and oncogenicity by showing that KSHV's ability to regulate HIF2α levels and localization to the endoplasmic reticulum (ER) in normoxia enables translation of viral lytic mRNAs through the HIF2α-regulated eIF4E2 translation-initiation complex. This mechanism of translation in infected cells is critical for lytic protein synthesis and contributes to KSHV-induced PDGFRA activation and VEGF secretion. Thus, KSHV regulation of the oxygen-sensing machinery allows virally infected cells to initiate translation via the mTOR-dependent eIF4E1 or the HIF2α-dependent, mTOR-independent, eIF4E2. This "translation initiation plasticity" (TRIP) is an oncoviral strategy used to optimize viral protein expression that links molecular strategies of viral replication to angiogenicity and oncogenesis.

The Polycomb protein RING1B enables estrogen-mediated gene expression by promoting enhancer-promoter interaction and R-loop formation.

Polycomb complexes have traditionally been prescribed roles as transcriptional repressors, though increasing evidence demonstrate they can also activate gene expression. However, the mechanisms underlying positive gene regulation mediated by Polycomb proteins are poorly understood. Here, we show that RING1B, a core component of Polycomb Repressive Complex 1, regulates enhancer-promoter interaction of the bona fide estrogen-activated GREB1 gene. Systematic characterization of RNA:DNA hybrid formation (R-loops), nascent transcription and RNA Pol II activity upon estrogen administration revealed a key role of RING1B in gene activation by regulating R-loop formation and RNA Pol II elongation. We also found that the estrogen receptor alpha (ERα) and RNA are both necessary for full RING1B recruitment to estrogen-activated genes. Notably, RING1B recruitment was mostly unaffected upon RNA Pol II depletion. Our findings delineate the functional interplay between RING1B, RNA and ERα to safeguard chromatin architecture perturbations required for estrogen-mediated gene regulation and highlight the crosstalk between steroid hormones and Polycomb proteins to regulate oncogenic programs.

Erratum: The uracil-DNA glycosylase UNG protects the fitness of normal and cancer B cells expressing AID.

[This corrects the article DOI: 10.1093/narcan/zcaa019.].

Trypanosoma cruzi Induces B Cells That Regulate the CD4+ T Cell Response.

Trypanosoma cruzi infection induces a polyclonal B cell proliferative response characterized by maturation to plasma cells, excessive generation of germinal centers, and secretion of parasite-unrelated antibodies. Although traditionally reduced to the humoral response, several infectious and non-infectious models revealed that B lymphocytes could regulate and play crucial roles in cellular responses. Here, we analyze the trypomastigote-induced effect on B cells, their effects on CD4+ T cells, and their correlation with in vivo findings. The trypomastigotes were able to induce the proliferation and the production of IL-10 or IL-6 of naïve B cells in co-culture experiments. Also, we found that IL-10-producing B220lo cells were elicited in vivo. We also found up-regulated expression of FasL and PD-L1, proteins involved in apoptosis induction and inhibition of TCR signaling, and of BAFF and APRIL mRNAs, two B-cell growth factors. Interestingly, it was observed that IL-21, which plays a critical role in regulatory B cell differentiation, was significantly increased in B220+/IL-21+ in in vivo infections. This is striking since the secretion of IL-21 is associated with T helper follicular cells. Furthermore, trypomastigote-stimulated B-cell conditioned medium dramatically reduced the proliferation and increased the apoptotic rate on CD3/CD28 activated CD4+ T cells, suggesting the development of effective regulatory B cells. In this condition, CD4+ T cells showed a marked decrease in proliferation and viability with marginal IL-2 or IFNγ secretion, which is counterproductive with an efficient immune response against T. cruzi. Altogether, our results show that B lymphocytes stimulated with trypomastigotes adopt a particular phenotype that exerts a strong regulation of this T cell compartment by inducing apoptosis, arresting cell division, and affecting the developing of a proinflammatory response.

Estrogen induces dynamic ERα and RING1B recruitment to control gene and enhancer activities in luminal breast cancer.

RING1B, a core Polycomb repressive complex 1 subunit, is a histone H2A ubiquitin ligase essential for development. RING1B is overexpressed in patients with luminal breast cancer (BC) and recruited to actively transcribed genes and enhancers co-occupied by the estrogen receptor α (ERα). Whether ERα-induced transcriptional programs are mediated by RING1B is not understood. We show that prolonged estrogen administration induces transcriptional output and chromatin landscape fluctuations. RING1B loss impairs full estrogen-mediated gene expression and chromatin accessibility for key BC transcription factors. These effects were mediated, in part, by RING1B enzymatic activity and nucleosome binding functions. RING1B is recruited in a cyclic manner to ERα, FOXA1, and GRHL2 cobound sites and regulates estrogen-induced enhancers and ERα recruitment. Last, ChIP exo revealed multiple binding events of these factors at single-nucleotide resolution, including RING1B occupancy approximately 10 base pairs around ERα bound sites. We propose RING1B as a key regulator of the dynamic, liganded-ERα transcriptional regulatory circuit in luminal BC.

The uracil-DNA glycosylase UNG protects the fitness of normal and cancer B cells expressing AID.

In B lymphocytes, the uracil N-glycosylase (UNG) excises genomic uracils made by activation-induced deaminase (AID), thus underpinning antibody gene diversification and oncogenic chromosomal translocations, but also initiating faithful DNA repair. Ung-/- mice develop B-cell lymphoma (BCL). However, since UNG has anti- and pro-oncogenic activities, its tumor suppressor relevance is unclear. Moreover, how the constant DNA damage and repair caused by the AID and UNG interplay affects B-cell fitness and thereby the dynamics of cell populations in vivo is unknown. Here, we show that UNG specifically protects the fitness of germinal center B cells, which express AID, and not of any other B-cell subset, coincident with AID-induced telomere damage activating p53-dependent checkpoints. Consistent with AID expression being detrimental in UNG-deficient B cells, Ung-/- mice develop BCL originating from activated B cells but lose AID expression in the established tumor. Accordingly, we find that UNG is rarely lost in human BCL. The fitness preservation activity of UNG contingent to AID expression was confirmed in a B-cell leukemia model. Hence, UNG, typically considered a tumor suppressor, acquires tumor-enabling activity in cancer cell populations that express AID by protecting cell fitness.

Interplay between HGAL and Grb2 proteins regulates B-cell receptor signaling.

Human germinal center (GC)-associated lymphoma (HGAL) is an adaptor protein expressed in GC B cells. HGAL regulates cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. Herein, we demonstrate phosphorylation of HGAL by Syk and Lyn kinases at tyrosines Y80, Y86, Y106Y107, Y128, and Y148. The HGAL YEN motif (amino acids 107-109) is similar to the phosphopeptide motif pYXN used as a binding site to the growth factor receptor-bound protein 2 (Grb2). We demonstrate by biochemical and molecular methodologies that HGAL directly interacts with Grb2. Concordantly, microscopy studies demonstrate HGAL-Grb2 colocalization in the membrane central supramolecular activation clusters (cSMAC) following BCR activation. Mutation of the HGAL putative binding site to Grb2 abrogates the interaction between these proteins. Further, this HGAL mutant localizes exclusively in the peripheral SMAC and decreases the rate and intensity of BCR accumulation in the cSMAC. Furthermore, we demonstrate that Grb2, HGAL, and Syk interact in the same complex, but Grb2 does not modulate the effects of HGAL on Syk kinase activity. Overall, the interplay between the HGAL and Grb2 regulates the magnitude of BCR signaling and synapse formation.

LMO2 Confers Synthetic Lethality to PARP Inhibition in DLBCL.

Deficiency in DNA double-strand break (DSB) repair mechanisms has been widely exploited for the treatment of different malignances, including homologous recombination (HR)-deficient breast and ovarian cancers. Here we demonstrate that diffuse large B cell lymphomas (DLBCLs) expressing LMO2 protein are functionally deficient in HR-mediated DSB repair. Mechanistically, LMO2 inhibits BRCA1 recruitment to DSBs by interacting with 53BP1 during repair. Similar to BRCA1-deficient cells, LMO2-positive DLBCLs and T cell acute lymphoblastic leukemia (T-ALL) cells exhibit a high sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors. Furthermore, chemotherapy and PARP inhibitors synergize to inhibit the growth of LMO2-positive tumors. Together, our results reveal that LMO2 expression predicts HR deficiency and the potential therapeutic use of PARP inhibitors in DLBCL and T-ALL.

PRMT5 Regulates DNA Repair by Controlling the Alternative Splicing of Histone-Modifying Enzymes.

Protein arginine methyltransferase 5 (PRMT5) is overexpressed in many cancer types and is a promising therapeutic target for several of them, including leukemia and lymphoma. However, we and others have reported that PRMT5 is essential for normal physiology. This dependence may become dose limiting in a therapeutic setting, warranting the search for combinatorial approaches. Here, we report that PRMT5 depletion or inhibition impairs homologous recombination (HR) DNA repair, leading to DNA-damage accumulation, p53 activation, cell-cycle arrest, and cell death. PRMT5 symmetrically dimethylates histone and non-histone substrates, including several components of the RNA splicing machinery. We find that PRMT5 depletion or inhibition induces aberrant splicing of the multifunctional histone-modifying and DNA-repair factor TIP60/KAT5, which selectively affects its lysine acetyltransferase activity and leads to impaired HR. As HR deficiency sensitizes cells to PARP inhibitors, we demonstrate here that PRMT5 and PARP inhibitors have synergistic effects on acute myeloid leukemia cells.

A licensing step links AID to transcription elongation for mutagenesis in B cells.

Activation-induced deaminase (AID) mutates the immunoglobulin (Ig) genes to initiate somatic hypermutation (SHM) and class switch recombination (CSR) in B cells, thus underpinning antibody responses. AID mutates a few hundred other loci, but most AID-occupied genes are spared. The mechanisms underlying productive deamination versus non-productive AID targeting are unclear. Here we show that three clustered arginine residues define a functional AID domain required for SHM, CSR, and off-target activity in B cells without affecting AID deaminase activity or Escherichia coli mutagenesis. Both wt AID and mutants with single amino acid replacements in this domain broadly associate with Spt5 and chromatin and occupy the promoter of AID target genes. However, mutant AID fails to occupy the corresponding gene bodies and loses association with transcription elongation factors. Thus AID mutagenic activity is determined not by locus occupancy but by a licensing mechanism, which couples AID to transcription elongation.

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Viral IL-6 (vIL-6) Enhances Immunoglobulin Class-Switch Recombination.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic gamma-herpesvirus that causes AIDS-associated Kaposi sarcoma (KS) and several lymphoproliferative disorders. During the humoral immune response antigen-activated mature B cells acquire functional diversification by immunoglobulin heavy chain (IgH) class-switch recombination (CSR). CSR is initiated by activation-induced cytidine deaminase (AID) which targets highly repetitive switch (S)-regions to mediate DNA double-stranded breaks (DSBs) in the IgH locus facilitating intramolecular recombination. Here we show that in the context of cytokine stimulation, CSR is enhanced in murine B cells exposed only to replication-competent KSHV in an environment of KSHV infection, which coincided with elevated AID transcripts. Using murine splenic B cells and the mouse lymphoma CH12F3-2 CSR system, we identified that vIL-6, but not murine IL-6, increased class-switching, which correlated with upregulated AID expression. Together, these data suggest a regulatory role for KSHV vIL-6 in functionally modulating B cell biology by promoting CSR, which may in part explain how KSHV infection influences humoral immunity and affect KSHV pathogenesis.

Epstein-Barr virus-positive follicular lymphoma.

Epstein-Barr virus (EBV) -associated follicular lymphoma is only rarely reported. Herein, we report the largest series analyzing prevalence and clinicopathologic characteristics of EBV-associated follicular lymphoma occurring in unselected cases. Out of 382 analyzed cases, 10 EBV-positive follicular lymphomas were identified (prevalence=2.6%, 95% confidence interval 1.3-4.0%). All EBV-positive follicular lymphomas showed EBV-encoded small RNA-positive lymphoma cells present in a follicular distribution. Of these, eight also had tissue available for testing of expression of latent membrane protein 1 (LMP1), out of which six (75%) were positive. There was a significant association with grades 3A-3B follicular lymphoma (P<0.0001) and CD30 expression (P=0.0002). EBV-positive follicular lymphomas were otherwise morphologically and immunophenotypically indistinguishable from EBV-negative cases of similar grade. Nine of the EBV-positive follicular lymphomas occurred in patients with no known history of immunosuppression, while one patient had a history of hydroxychloroquine administration for Sjögren's syndrome. The mean age in the EBV-positive and -negative follicular lymphomas was 56 (range 31-83 years) and 49 years (range 25-92 years), respectively, with no statistically significant difference. Seven of the patients with EBV-positive follicular lymphoma had additional biopsies from different time points available for review, all of which showed progression of disease in the form of progression of tumor grade. Five of these progressed to diffuse large B-cell lymphoma, one of which had tissue available for testing and was EBV-positive. Our findings suggest that EBV infection may have a role in lymphomagenesis and/or disease progression in a subset of follicular lymphomas, thereby expanding the spectrum of recognized EBV-associated B-cell lymphomas.