Intra-tumoral and peripheral blood TIGIT and PD-1 as immune biomarkers in nodular lymphocyte predominant Hodgkin lymphoma.

In classical Hodgkin lymphoma (cHL), responsiveness to immune-checkpoint blockade (ICB) is associated with specific tumor microenvironment (TME) and peripheral blood features. The role of ICB in nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is not established. To gain insights into its potential in NLPHL, we compared TME and peripheral blood signatures between HLs using an integrative multiomic analysis. A discovery/validation approach in 121 NLPHL and 114 cHL patients highlighted >2-fold enrichment in programmed cell death-1 (PD-1) and T-cell Ig and ITIM domain (TIGIT) gene expression for NLPHL versus cHL. Multiplex imaging showed marked increase in intra-tumoral protein expression of PD-1+ (and/or TIGIT+) CD4+ T-cells and PD-1+CD8+ T-cells in NLPHL compared to cHL. This included T-cells that rosetted with lymphocyte predominant (LP) and Hodgkin Reed-Sternberg (HRS) cells. In NLPHL, intra-tumoral PD-1+CD4+ T-cells frequently expressed TCF-1, a marker of heightened T-cell response to ICB. The peripheral blood signatures between HLs were also distinct, with higher levels of PD-1+TIGIT+ in TH1, TH2, and regulatory CD4+ T-cells in NLPHL versus cHL. Circulating PD-1+CD4+ had high levels of TCF-1. Notably, in both lymphomas, highly expanded populations of clonal TIGIT+PD-1+CD4+ and TIGIT+PD-1+CD8+ T-cells in the blood were also present in the TME, indicating that immune-checkpoint expressing T-cells circulated between intra-tumoral and blood compartments. In in vitro assays, ICB was capable of reducing rosette formation around LP and HRS cells, suggesting that disruption of rosetting may be a mechanism of action of ICB in HL. Overall, results indicate that further evaluation of ICB is warranted in NLPHL.

Identification of WNK1 as a therapeutic target to suppress IgH/MYC expression in multiple myeloma.

Multiple myeloma (MM) remains an incurable hematological malignancy demanding innovative therapeutic strategies. Targeting MYC, the notorious yet traditionally undruggable oncogene, presents an appealing avenue. Here, using a genome-scale CRISPR-Cas9 screen, we identify the WNK lysine-deficient protein kinase 1 (WNK1) as a regulator of MYC expression in MM cells. Genetic and pharmacological inhibition of WNK1 reduces MYC expression and, further, disrupts the MYC-dependent transcriptional program. Mechanistically, WNK1 inhibition attenuates the activity of the immunoglobulin heavy chain (IgH) enhancer, thus reducing MYC transcription when this locus is translocated near the MYC locus. WNK1 inhibition profoundly impacts MM cell behaviors, leading to growth inhibition, cell-cycle arrest, senescence, and apoptosis. Importantly, the WNK inhibitor WNK463 inhibits MM growth in primary patient samples as well as xenograft mouse models and exhibits synergistic effects with various anti-MM compounds. Collectively, our study uncovers WNK1 as a potential therapeutic target in MM.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

EZH2 inhibition reactivates epigenetically silenced FMR1 and normalizes molecular and electrophysiological abnormalities in fragile X syndrome neurons.

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here, using a candidate-based shRNA screen, we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1- SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells, neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2, for which an FDA-approved small molecule inhibitor, EPZ6438 (also known as tazemetostat), is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately, EZH2 inhibitors do not efficiently cross the blood-brain barrier, limiting their therapeutic use for FXS. Recently, antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons, and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively, our results establish EZH2 inhibition in general, and EZH2 ASOs in particular, as a therapeutic approach for FXS.

ARID1A orchestrates SWI/SNF-mediated sequential binding of transcription factors with ARID1A loss driving pre-memory B cell fate and lymphomagenesis.

ARID1A, a subunit of the canonical BAF nucleosome remodeling complex, is commonly mutated in lymphomas. We show that ARID1A orchestrates B cell fate during the germinal center (GC) response, facilitating cooperative and sequential binding of PU.1 and NF-kB at crucial genes for cytokine and CD40 signaling. The absence of ARID1A tilts GC cell fate toward immature IgM+CD80-PD-L2- memory B cells, known for their potential to re-enter new GCs. When combined with BCL2 oncogene, ARID1A haploinsufficiency hastens the progression of aggressive follicular lymphomas (FLs) in mice. Patients with FL with ARID1A-inactivating mutations preferentially display an immature memory B cell-like state with increased transformation risk to aggressive disease. These observations offer mechanistic understanding into the emergence of both indolent and aggressive ARID1A-mutant lymphomas through the formation of immature memory-like clonal precursors. Lastly, we demonstrate that ARID1A mutation induces synthetic lethality to SMARCA2/4 inhibition, paving the way for potential precision therapy for high-risk patients.

SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.

SMARCA4 encodes one of two mutually exclusive ATPase subunits in the BRG/BRM associated factor (BAF) complex that is recruited by transcription factors (TFs) to drive chromatin accessibility and transcriptional activation. SMARCA4 is among the most recurrently mutated genes in human cancer, including ∼30% of germinal center (GC)-derived Burkitt lymphomas. In mice, GC-specific Smarca4 haploinsufficiency cooperated with MYC over-expression to drive lymphomagenesis. Furthermore, monoallelic Smarca4 deletion drove GC hyperplasia with centroblast polarization via significantly increased rates of centrocyte recycling to the dark zone. Mechanistically, Smarca4 loss reduced the activity of TFs that are activated in centrocytes to drive GC-exit, including SPI1 (PU.1), IRF family, and NF-κB. Loss of activity for these factors phenocopied aberrant BCL6 activity within murine centrocytes and human Burkitt lymphoma cells. SMARCA4 therefore facilitates chromatin accessibility for TFs that shape centrocyte trajectories, and loss of fine-control of these programs biases toward centroblast cell-fate, GC hyperplasia and lymphoma.

Inhibition of MALT1 and BCL2 Induces Synergistic Antitumor Activity in Models of B-Cell Lymphoma.

The activated B cell (ABC) subset of diffuse large B-cell lymphoma (DLBCL) is characterized by chronic B-cell receptor signaling and associated with poor outcomes when treated with standard therapy. In ABC-DLBCL, MALT1 is a core enzyme that is constitutively activated by stimulation of the B-cell receptor or gain-of-function mutations in upstream components of the signaling pathway, making it an attractive therapeutic target. We discovered a novel small-molecule inhibitor, ABBV-MALT1, that potently shuts down B-cell signaling selectively in ABC-DLBCL preclinical models leading to potent cell growth and xenograft inhibition. We also identified a rational combination partner for ABBV-MALT1 in the BCL2 inhibitor, venetoclax, which when combined significantly synergizes to elicit deep and durable responses in preclinical models. This work highlights the potential of ABBV-MALT1 monotherapy and combination with venetoclax as effective treatment options for patients with ABC-DLBCL.

Comprehensive characterization of IFNγ signaling in acute myeloid leukemia reveals prognostic and therapeutic strategies.

Interferon gamma (IFNγ) is a critical cytokine known for its diverse roles in immune regulation, inflammation, and tumor surveillance. However, while IFNγ levels were elevated in sera of most newly diagnosed acute myeloid leukemia (AML) patients, its complex interplay in AML remains insufficiently understood. We aim to characterize these complex interactions through comprehensive bulk and single-cell approaches in bone marrow of newly diagnosed AML patients. We identify monocytic AML as having a unique microenvironment characterized by IFNγ producing T and NK cells, high IFNγ signaling, and immunosuppressive features. IFNγ signaling score strongly correlates with venetoclax resistance in primary AML patient cells. Additionally, IFNγ treatment of primary AML patient cells increased venetoclax resistance. Lastly, a parsimonious 47-gene IFNγ score demonstrates robust prognostic value. In summary, our findings suggest that inhibiting IFNγ is a potential treatment strategy to overcoming venetoclax resistance and immune evasion in AML patients.