A multiprotein supercomplex controlling oncogenic signalling in lymphoma.

B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCß to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.

A novel model of controlling PD-L1 expression in ALK+ anaplastic large cell lymphoma revealed by CRISPR screening.

The success of programmed cell death protein 1 (PD-1)/PD-L1-based immunotherapy highlights the critical role played by PD-L1 in cancer progression and reveals an urgent need to develop new approaches to attenuate PD-L1 function by gaining insight into how its expression is controlled. Anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALK+ ALCL) expresses a high level of PD-L1 as a result of the constitutive activation of multiple oncogenic signaling pathways downstream of ALK activity, making it an excellent model in which to define the signaling processes responsible for PD-L1 upregulation in tumor cells. Here, using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 library screening, we sought a comprehensive understanding of the molecular effectors required for PD-L1 regulation in ALK+ ALCL. Indeed, we determined that PD-L1 induction is dependent on the nucleophosmin-ALK oncoprotein activation of STAT3, as well as a signalosome containing GRB2/SOS1, which activates the MEK-ERK and PI3K-AKT signaling pathways. These signaling networks, through STAT3 and the GRB2/SOS1, ultimately induce PD-L1 expression through the action of transcription factors IRF4 and BATF3 on the enhancer region of the PD-L1 gene. IRF4 and BATF3 are essential for PD-L1 upregulation, and IRF4 expression is correlated with PD-L1 levels in primary ALK+ ALCL tissues. Targeting this oncogenic signaling pathway in ALK+ ALCL largely inhibited the ability of PD-L1-mediated tumor immune escape when cocultured with PD-1-positive T cells and natural killer cells. Thus, our identification of this previously unrecognized regulatory hub not only accelerates our understanding of the molecular circuitry that drives tumor immune escape but also provides novel opportunities to improve immunotherapeutic intervention strategies.

Regulation of normal B-cell differentiation and malignant B-cell survival by OCT2.

The requirement for the B-cell transcription factor OCT2 (octamer-binding protein 2, encoded by Pou2f2) in germinal center B cells has proved controversial. Here, we report that germinal center B cells are formed normally after depletion of OCT2 in a conditional knockout mouse, but their proliferation is reduced and in vivo differentiation to antibody-secreting plasma cells is blocked. This finding led us to examine the role of OCT2 in germinal center-derived lymphomas. shRNA knockdown showed that almost all diffuse large B-cell lymphoma (DLBCL) cell lines are addicted to the expression of OCT2 and its coactivator OCA-B. Genome-wide chromatin immunoprecipitation (ChIP) analysis and gene-expression profiling revealed the broad transcriptional program regulated by OCT2 that includes the expression of STAT3, IL-10, ELL2, XBP1, MYC, TERT, and ADA. Importantly, genetic alteration of OCT2 is not a requirement for cellular addiction in DLBCL. However, we detected amplifications of the POU2F2 locus in DLBCL tumor biopsies and a recurrent mutation of threonine 223 in the DNA-binding domain of OCT2. This neomorphic mutation subtly alters the DNA-binding preference of OCT2, leading to the transactivation of noncanonical target genes including HIF1a and FCRL3 Finally, by introducing mutations designed to disrupt the OCT2-OCA-B interface, we reveal a requirement for this protein-protein interface that ultimately might be exploited therapeutically. Our findings, combined with the predominantly B-cell-restricted expression of OCT2 and the absence of a systemic phenotype in our knockout mice, suggest that an OCT2-targeted therapeutic strategy would be efficacious in both major subtypes of DLBCL while avoiding systemic toxicity.

DNMT1 maintains progenitor function in self-renewing somatic tissue.

Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation. Furthermore, UHRF1 (refs 9, 10), a component of the DNA methylation machinery that targets DNMT1 to hemi-methylated DNA, is also necessary to suppress premature differentiation and sustain proliferation. In contrast, Gadd45A and B, which promote active DNA demethylation, are required for full epidermal differentiation gene induction. These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.

Overcoming Acquired Epigenetic Resistance to BTK Inhibitors.

The use of Bruton tyrosine kinase (BTK) inhibitors to block B-cell receptor (BCR)-dependent NF-κB activation in lymphoid malignancies has been a major clinical advance, yet acquired therapeutic resistance is a recurring problem. We modeled the development of resistance to the BTK inhibitor ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. The resultant phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in the activation of phospholipase Cγ2, thereby sustaining NF-κB activity. The interaction of RAC2 with phospholipase Cγ2 was also increased in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTK inhibitor treatment. We identified clinically available drugs that can treat epigenetic ibrutinib resistance, suggesting combination therapeutic strategies. SIGNIFICANCE: In diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought.See related commentary by Pasqualucci, p. 555. This article is highlighted in the In This Issue feature, p. 549.

KIR receptor-ligand incompatibility predicts killing of osteosarcoma cell lines by allogeneic NK cells.

BACKGROUND: The effectiveness of killer immunoglobulin-like receptor (KIR) incompatible, alloreactive natural killer (NK) cells has been primarily documented in hematological malignancies following stem-cell transplant. This effect has not been thoroughly evaluated for pediatric solid tumors. In this study, we evaluated KIR receptor-ligand incompatibility of NK cells against osteosarcoma cell lines. PROCEDURE: Following the KIR receptor-ligand mismatch model, MHC I cell surface expression and KIR ligand mRNA content of 3 osteosarcoma cell lines was determined by flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively. NK cells were isolated from healthy volunteer donor peripheral blood mononuclear cells (PBMCs) and KIR surface expression determined by flow cytometry. An Annexin-V based flow cytometric killing assay was used to determine % of dying osteosarcoma target cells by donor NK effector cells. RESULTS: One of seven healthy volunteer donors tested lacked phenotypic expression of one KIR. However, variable expression of KIR ligands was observed in 3 osteosarcoma cell lines. The highest rates of dying cells were seen in osteosarcoma cells with the lowest KIR ligand expression. Following down-regulation of KIR ligand expression, an increased susceptibility to NK cell-mediated killing was observed in a previously NK-resistant osteosarcoma cell line. CONCLUSIONS: Variable MHC I and KIR ligand expression was observed in osteosarcoma cell lines and this resulted in variable susceptibility to NK cell-mediated killing predicted by the degree of KIR receptor-ligand incompatibility. Collectively, these data provide rationale for the study of KIR incompatible stem-cell transplant for osteosarcoma, although further studies with fresh osteosarcoma samples are necessary.

Protocols for CRISPR-Cas9 Screening in Lymphoma Cell Lines.

Genome-wide screens are a powerful technique to dissect the complex network of genes regulating diverse cellular phenotypes. The recent adaptation of the CRISPR-Cas9 system for genome engineering has revolutionized functional genomic screening. Here, we present protocols used to introduce Cas9 into human lymphoma cell lines, produce high-titer lentivirus of a genome-wide sgRNA library, transduce and culture cells during the screen, isolate genomic DNA, and prepare a custom library for next-generation sequencing. These protocols were tailored for loss-of-function CRISPR screens in human lymphoma cell lines but are highly amenable for other experimental purposes.

Targeting the HTLV-I-Regulated BATF3/IRF4 Transcriptional Network in Adult T Cell Leukemia/Lymphoma.

Adult T cell leukemia/lymphoma (ATLL) is a frequently incurable disease associated with the human lymphotropic virus type I (HTLV-I). RNAi screening of ATLL lines revealed that their proliferation depends on BATF3 and IRF4, which cooperatively drive ATLL-specific gene expression. HBZ, the only HTLV-I encoded transcription factor that is expressed in all ATLL cases, binds to an ATLL-specific BATF3 super-enhancer and thereby regulates the expression of BATF3 and its downstream targets, including MYC. Inhibitors of bromodomain-and-extra-terminal-domain (BET) chromatin proteins collapsed the transcriptional network directed by HBZ and BATF3, and were consequently toxic for ATLL cell lines, patient samples, and xenografts. Our study demonstrates that the HTLV-I oncogenic retrovirus exploits a regulatory module that can be attacked therapeutically with BET inhibitors.

CSNK1a1 Regulates PRMT1 to Maintain the Progenitor State in Self-Renewing Somatic Tissue.

Somatic progenitors sustain tissue self-renewal while suppressing premature differentiation. Protein arginine methyltransferases (PRMTs) affect many processes; however, their role in progenitor function is incompletely understood. PRMT1 was found to be the most highly expressed PRMT in epidermal progenitors and the most downregulated PRMT during differentiation. In targeted mouse knockouts and in long-term regenerated human mosaic epidermis in vivo, epidermal PRMT1 loss abolished progenitor self-renewal and led to premature differentiation. Mass spectrometry of the PRMT1 protein interactome identified the CSNK1a1 kinase, which also proved essential for progenitor maintenance. CSNK1a1 directly bound and phosphorylated PRMT1 to control its genomic targeting to PRMT1-sustained proliferation genes as well as PRMT1-suppressed differentiation genes. Among the latter were GRHL3, whose derepression was required for the premature differentiation seen with PRMT1 and CSNK1a1 loss. Maintenance of the progenitors thus requires cooperation by PRMT1 and CSNK1a1 to sustain proliferation gene expression and suppress premature differentiation driven by GRHL3.

Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3.

The recent discovery of H3K27me3 demethylases suggests that H3K27me3 may dynamically regulate gene expression, but this potential role in mammalian tissue homeostasis remains uncharacterized. In the epidermis, a tissue that balances stem cell self-renewal with differentiation, H3K27me3, occupies the promoters of many differentiation genes. During calcium-induced differentiation, H3K27me3 was erased at these promoters in concert with loss of PcG protein occupancy and increased binding by the H3K27me3 demethylase, JMJD3. Within epidermal tissue, JMJD3 depletion blocked differentiation, while active JMJD3 dominantly induced it. These results indicate that epigenetic derepression by JMJD3 controls mammalian epidermal differentiation.