ETS1 phosphorylation at threonine 38 is associated with the cell of origin of diffuse large B cell lymphoma and sustains the growth of tumour cells.

The transcriptional factor ETS1 is upregulated in 25% of diffuse large B cell lymphoma (DLBCL). Here, we studied the role of ETS1 phosphorylation at threonine 38, a marker for ETS1 activation, in DLBCL cellular models and clinical specimens. p-ETS1 was detected in activated B cell-like DLBCL (ABC), not in germinal centre B-cell-like DLBCL (GCB) cell lines and, accordingly, it was more common in ABC than GCB DLBCL diagnostic biopsies. MEK inhibition decreased both baseline and IgM stimulation-induced p-ETS1 levels. Genetic inhibition of phosphorylation of ETS1 at threonine 38 affected the growth and the BCR-mediated transcriptome program in DLBCL cell lines. Our data demonstrate that ETS1 phosphorylation at threonine 38 is important for the growth of DLBCL cells and its pharmacological inhibition could benefit lymphoma patients.

PAK Kinase Inhibition Has Therapeutic Activity in Novel Preclinical Models of Adult T-Cell Leukemia/Lymphoma.

PURPOSE: To evaluate therapeutic activity of PAK inhibition in ATLL and to characterize the role of PAK isoforms in cell proliferation, survival, and adhesion of ATLL cells in preclinical models. EXPERIMENTAL DESIGN: Frequency and prognostic impact of PAK2 amplification were evaluated in an ATLL cohort of 370 cases. Novel long-term cultures and in vivo xenograft models were developed using primary ATLL cells from North American patients. Two PAK inhibitors were used to block PAK kinase activity pharmacologically. siRNA-based gene silencing approach was used to genetically knockdown (KD) PAK1 and PAK2 in ATLL cell lines. RESULTS: PAK1/2/4 are the three most abundantly expressed PAK family members in ATLL. PAK2 amplifications are seen in 24% of ATLLs and are associated with worse prognosis in a large patient cohort. The pan-PAK inhibitor PF-3758309 (PF) has strong in vitro and in vivo activity in a variety of ATLL preclinical models. These activities of PF are likely attributed to its ability to target several PAK isoforms simultaneously because genetic silencing of either PAK1 or PAK2 produced more modest effects. PAK2 plays a major role in CADM1-mediated stromal interaction, which is an important step in systemic dissemination of the disease. This finding is consistent with the observation that PAK2 amplification is more frequent in aggressive ATLLs and correlates with inferior outcome. CONCLUSIONS: PAK2, a gene frequently amplified in ATLL, facilitates CADM1-mediated stromal interaction and promotes survival of ATLL cells. Taken together, PAK inhibition may hold significant promise as a targeted therapy for aggressive ATLLs.

North American ATLL has a distinct mutational and transcriptional profile and responds to epigenetic therapies.

Adult T-cell leukemia lymphoma (ATLL) is a rare T cell neoplasm that is endemic in Japanese, Caribbean, and Latin American populations. Most North American ATLL patients are of Caribbean descent and are characterized by high rates of chemo-refractory disease and worse prognosis compared with Japanese ATLL. To determine genomic differences between these 2 cohorts, we performed targeted exon sequencing on 30 North American ATLL patients and compared the results with the Japanese ATLL cases. Although the frequency of TP53 mutations was comparable, the mutation frequency in epigenetic and histone modifying genes (57%) was significantly higher, whereas the mutation frequency in JAK/STAT and T-cell receptor/NF-κB pathway genes was significantly lower. The most common type of epigenetic mutation is that affecting EP300 (20%). As a category, epigenetic mutations were associated with adverse prognosis. Dissimilarities with the Japanese cases were also revealed by RNA sequencing analysis of 9 primary patient samples. ATLL samples with a mutated EP300 gene have decreased total and acetyl p53 protein and a transcriptional signature reminiscent of p53-mutated cancers. Most importantly, decitabine has highly selective single-agent activity in the EP300-mutated ATLL samples, suggesting that decitabine treatment induces a synthetic lethal phenotype in EP300-mutated ATLL cells. In conclusion, we demonstrate that North American ATLL has a distinct genomic landscape that is characterized by frequent epigenetic mutations that are targetable preclinically with DNA methyltransferase inhibitors.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.

UNLABELLED: The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE: CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

CD19 is a major B cell receptor-independent activator of MYC-driven B-lymphomagenesis.

PAX5, a B cell-specific transcription factor, is overexpressed through chromosomal translocations in a subset of B cell lymphomas. Previously, we had shown that activation of immunoreceptor tyrosine-based activation motif (ITAM) proteins and B cell receptor (BCR) signaling by PAX5 contributes to B-lymphomagenesis. However, the effect of PAX5 on other oncogenic transcription factor-controlled pathways is unknown. Using a MYC-induced murine lymphoma model as well as MYC-transformed human B cell lines, we found that PAX5 controls c-MYC protein stability and steady-state levels. This promoter-independent, posttranslational mechanism of c-MYC regulation was independent of ITAM/BCR activity. Instead it was controlled by another PAX5 target, CD19, through the PI3K-AKT-GSK3ß axis. Consequently, MYC levels in B cells from CD19-deficient mice were sharply reduced. Conversely, reexpression of CD19 in murine lymphomas with spontaneous silencing of PAX5 boosted MYC levels, expression of its key target genes, cell proliferation in vitro, and overall tumor growth in vivo. In human B-lymphomas, CD19 mRNA levels were found to correlate with those of MYC-activated genes. They also negatively correlated with the overall survival of patients with lymphoma in the same way that MYC levels do. Thus, CD19 is a major BCR-independent regulator of MYC-driven neoplastic growth in B cell neoplasms.

c-Myb oncoprotein is an essential target of the dleu2 tumor suppressor microRNA cluster.

The dleu2 tumor suppressor locus encodes two microRNAs, miR-15a and miR-16, which are thought to play an important role in B-cell neoplasms. However, relatively little is known about proteins that regulate or are regulated by this microRNA cluster. Here we demonstrate that the Pax5 oncoprotein downregulates the dleu2 gene and at the same time boosts expression of its own heterodimeric partner c-Myb. Interestingly, c-Myb upregulation occurs primarily at a post-transcriptional level, suggesting that it might be a target for microRNAs such as miR-15a/16. Indeed, miR-15a/16 have predicted binding sites in the c-Myb 3'-UTR and through them diminish protein output in luciferase sensor assays. Moreover, forced overexpression of miR-15a/16 reduces endogenous c-Myb levels and compromises Pax5 function. Conversely, restoration of c-Myb levels partly alleviates tumors suppressive effects of miR-15a/16, suggesting that c-Myb is a key downstream target of this microRNA cluster.

Interleukin-10 expression in macrophages during phagocytosis of apoptotic cells is mediated by homeodomain proteins Pbx1 and Prep-1.

Production of interleukin (IL)-10, a major immunoregulatory cytokine, by phagocytes during clearance of apoptotic cells is critical to ensuring cellular homeostasis and suppression of autoimmunity. Little is known about the regulatory mechanisms in this fundamental process. We report that IL-10 production stimulated by apoptotic cells was regulated at the point of transcription in a manner dependent on p38 mitogen-activated protein kinase, partially on the scavenger receptor CD36, and required cell-cell contact but not phagocytosis. By using a reporter assay, we mapped the apoptotic-cell-response element (ACRE) in the human IL10 promoter and provide biochemical and physiological evidence that ACRE mediates the transcriptional activation of IL10 by pre-B cell leukemia transcription factor-1b and another Hox cofactor Pbx-regulating protein 1 in response to apoptotic cells. This study establishes a role of two developmentally critical factors (Pbx1 and Prep-1) in the regulation of homeostasis in the immune system.

Regulation of cytokine production during phagocytosis of apoptotic cells.

Loss of self-tolerance and expansion of auto-reactive lymphocytes are the basis for autoimmunity. Apoptosis and the rapid clearance of apoptotic cells by phagocytes usually occur as coordinated processes that ensure regulated cellularity and stress response with non-pathological outcomes. Defects in clearance of apoptotic cells would contribute to the generation of self-reactive lymphocytes, which drive autoimmune disorders such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The IL-12 family of cytokines (IL-12, IL-23, and IL-27) and IL-10 are produced by phagocytic macrophages and play critical roles in the regulation of antigen-presenting cells (APCs) and effector lymphocytes during an immune response to pathogens. Inappropriate expression of these cytokines and their dysregulated activities have been strongly implicated in the pathogenesis of several autoimmune diseases. The production of pro- and anti-inflammatory cytokines by phagocytic APCs is delicately regulated during the ingestion of apoptotic cells as part of an intrinsic mechanism to prevent inflammatory autoimmune reactions. How apoptotic cell-derived signals regulate cytokine production is poorly understood. A recent study by our group demonstrated that phagocytosis of apoptotic cells by activated macrophages results in strong inhibition of IL-12 p35 gene expression by activating a novel transcription repressor, which we named GC-binding protein (GC-BP), through tyrosine dephosphorylation. We are also beginning to understand the molecular mechanisms underlying apoptotic cell-triggered production of IL-10 by phagocytes. These studies will help to elucidate some novel immune regulatory mechanisms and explore the regulation of immune responses to autoantigens with potentials to discover new therapeutic targets for the treatment of autoimmune disorders.

Immunological consequences of macrophage-mediated clearance of apoptotic cells.

Apoptosis and the rapid clearance of apoptotic cells by professional or nonprofessional phagocytes are normal and coordinated processes that ensure controlled cell growth with a nonpathological outcome. Defects in clearance of apoptotic cells by macrophages have serious consequences often resulting in autoimmune disorders. Phagocyte-derived immunoregulatory cytokines such as Interleukin-12 and Interleukin-10 play pivotal roles in the etiology and pathology of many autoimmune diseases. Elucidation of the apoptotic cell-mediated signaling mechanisms involved in the control of pro- and anti-inflammatory cytokines during cell turnovers under normal and pathological conditions may help us counter the cytokine dysregulation and control inappropriate host immune reactions in pathological situations such as autoimmunity, infectious diseases, graft-versus-host disease, and cancer.

Interleukin-12: an update on its immunological activities, signaling and regulation of gene expression.

Interleukin-12 (IL-12) is a heterodimeric cytokine composed of the p35 and p40 subunits. It is produced by antigen-presenting cells and plays a critical role in host defense against intracellular microbial infection and control of malignancy via its ability to stimulate both innate and adaptive immune effector cells. The potency of IL-12 renders itself to stringent regulation of the timing, locality and magnitude of its production during an immune response. Subversion of the delicate control and balance frequently leads to immunologic disorders. In this article, we provide an update, since our last review of the subject four years ago, on recent advances in: (1) uncovering of novel activities of IL-12 and related molecules in various immunological settings and models; and (2) dissection of the physiological pathways involved in the modulation of IL-12 production by pathogens and immune regulators. The increased understanding of IL-12 immunobiology and expression will likely benefit the development of therapeutic modalities to correct immune dysfunctions.

PRAM-1 is required for optimal integrin-dependent neutrophil function.

PML-retinoic acid receptor alpha (RARalpha) regulated adaptor molecule 1 (PRAM-1) is an intracellular adaptor molecule that is upregulated during the induced granulocytic differentiation of promyelocytic leukemic cells and during normal human myelopoiesis. This report describes the generation of PRAM-1-deficient mice and an analysis of the function of this adaptor in neutrophil differentiation and mature neutrophil function. We demonstrate here that neutrophil differentiation is not impaired in PRAM-1-deficient mice and that PRAM-1-deficient neutrophils function normally following engagement of Fcgamma receptors. In contrast, mature PRAM-1-null neutrophils exhibit significant defects in adhesion-dependent reactive oxygen intermediate production and degranulation. Surprisingly, other integrin-dependent responses, such as cell spreading and activation of several signaling pathways, are normal. Together, these findings demonstrate the uncoupling of key integrin-dependent responses in the absence of PRAM-1 and show this adaptor to be critical for select integrin functions in neutrophils.