Eva-Maria Piskor

Activation-induced deaminase (AID) drives antibody diversification through class-switch recombination and somatic hypermutation of the immunoglobulin (Ig) genes, but its off-target mutagenic activity contributes to B cell lymphoma^1,2,3,4,5. How AID selectively mutates Ig loci and a restricted set of other genes remains unknown. Transcription is required for AID activity, but most transcribed genes are not mutated^1,5,6,7, and AID occupies more loci than it mutates^7,8,9. Here we identify the super elongation complex histone readers MLLT1 and MLLT3 as determinants of selective AID activity. Combined loss of MLLT1 and MLLT3 abolishes all AID-dependent mutagenic processes, recapitulating AID deficiency. AID-mutated genomic regions in mouse and human B cells are precisely marked by high MLLT1 and MLLT3 occupancy (MLLT1/MLLT3^high). Transcriptional changes after deleting both readers are modest and cannot explain the AID activity loss. Mechanistically, MLLT1 and MLLT3 are dispensable for global AID chromatin tethering but locally enrich AID downstream from promoter regions. Both readers bind to AID and act redundantly, but MLLT1 has a dominant role in mice through its intrinsically disordered region, which promotes condensates that selectively concentrate AID. Fusing AID to MLLT1 or MLLT3 is sufficient to restore class-switch recombination and mutagenesis in Mllt1^−/−Mllt3^−/− cells. These findings reveal that local MLLT1/MLLT3^high-dependent enrichment licenses AID at a restricted subset of genomic regions by spatially confining and concentrating its activity, probably through condensate formation.

Acute myeloid leukemia (AML) with rearrangement of the mixed lineage leukemia gene expresses MLL-AF9 fusion protein, a transcription factor that impairs differentiation and drives expansion of leukemic cells. In this work, the zinc finger protein “growth factor independent 1” together with the histone methyltransferase LSD1 is revealed to occupy the promoter and regulate the expression of the lncRNA ELDR (EGFR [epidermal growth factor receptor] long non-coding downstream RNA) in the rearranged Mixed Lineage Leukemia (MLL) (MLL-r) AML cell line THP-1. Forced ELDR overexpression enhanced the growth inhibition of an Lysine-Specific Demethylase 1 inhibitor (LSD1i)/all-trans retinoic acid (ATRA) combination treatment and reduced the capacity of these cells to generate leukemia in xenografts, leading to a longer leukemia-free survival. ELDR is found to bind the clamp protein Proliferating Cell Nuclear Antigen (PCNA) and the MCM5 helicases causing defects of DNA replication fork progression. Moreover, AML cells overexpressing ELDR had reduced chromatin accessibility and transcription at α-satellite repeats in centromeres. In addition, ELDR RNA was detected close to MLL-AF9 at centromeres suggesting that it impedes leukemic progression preferentially of MLL-r AML by interfering with both DNA replication and centromeric transcription. Our findings reveal novel functions of the lncRNA ELDR in DNA replication and centromere biology when expressed at high levels in AML cells with MLL rearrangements. These discoveries could provide rationale for future strategies to treat MLL-r AML, which has a poor prognosis in children and adults. Delivery of the ELDR RNA could potentially be used as an adjunct to LSD1i/ATRA treatment or other currently used chemotherapeutic drugs to develop novel therapies for these AML subtypes.

Oncogenic overexpression of MYC leads to the fatal deregulation of signaling pathways, cellular metabolism, and cell growth. MYC rearrangements are found frequently among non-Hodgkin B-cell lymphomas enforcing MYC overexpression. Genetically engineered mouse models (GEMMs) were developed to understand MYC-induced B-cell lymphomagenesis. Here, we highlight the advantages of using Eµ-Myc transgenic mice. We thoroughly compiled the available literature to discuss common challenges when using such mouse models. Furthermore, we give an overview of pathways affected by MYC based on knowledge gained from the use of GEMMs. We identified top regulators of MYC-induced lymphomagenesis, including some candidates that are not pharmacologically targeted yet.

Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC-induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC-overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.

Aging of the immune system is described as a progressive loss of the ability to respond to immunologic stimuli and is commonly referred to as immunosenescence. B cell immunosenescence is characterized by a decreased differentiation rate in the bone marrow and accumulation of antigen-experienced and age-associated B cells in secondary lymphoid organs (SLOs). A specific deletion of the POZ-domain of the transcription factor Miz-1 in pro-B cells, which is known to be involved in bone marrow hematopoiesis, leads to premature aging of the B cell lineage. In mice, this causes a severe reduction in bone marrow-derived B cells with a drastic decrease from the pre-B cell stage on. Further, mature, naïve cells in SLOs are reduced at an early age, while post-activation-associated subpopulations increase prematurely. We propose that Miz-1 interferes at several key regulatory checkpoints, critical during B cell aging, and counteracts a premature loss of immunocompetence. This enables the use of our mouse model to gain further insights into mechanisms of B cell aging and it can significantly contribute to understand molecular causes of impaired adaptive immune responses to counteract loss of immunocompetence and restore a functional immune response in the elderly.

Overexpression of MYC is a genuine cancer driver in lymphomas and related to poor prognosis. However, therapeutic targeting of the transcription factor MYC remains challenging. Here, we show that inhibition of the histone deacetylase 6 (HDAC6) using the HDAC6 inhibitor Marbostat-100 (M-100) reduces oncogenic MYC levels and prevents lymphomagenesis in a mouse model of MYC -induced aggressive B-cell lymphoma. M-100 specifically alters protein-protein interactions by switching the acetylation state of HDAC6 substrates, such as tubulin. Tubulin facilitates nuclear import of MYC, and MYC-dependent B-cell lymphoma cells rely on continuous import of MYC due to its high turn-over. Acetylation of tubulin impairs this mechanism and enables proteasomal degradation of MYC. M-100 targets almost exclusively B-cell lymphoma cells with high levels of MYC whereas non-tumor cells are not affected. M-100 induces massive apoptosis in human and murine MYC -overexpressing B-cell lymphoma cells. We identified the heat-shock protein DNAJA3 as an interactor of tubulin in an acetylation-dependent manner and overexpression of DNAJA3 resulted in a pronounced degradation of MYC. We propose a mechanism by which DNAJA3 associates with hyperacetylated tubulin in the cytoplasm to control MYC turnover. Taken together, our data demonstrate a beneficial role of HDAC6 inhibition in MYC-dependent B-cell lymphoma.