Sex-dependent Prognosis of Patients with Advanced Soft Tissue Sarcoma.

PURPOSE: To examine whether overall survival (OS) differs for male and female patients with advanced soft-tissue sarcoma (STS). EXPERIMENTAL DESIGN: The study included patients from Kaiser Permanente Northern California and Stanford Cancer Center with grade 2 and 3 locally advanced or metastatic STS whose tumor underwent next-generation sequencing. We used Cox regression modeling to examine association of sex and OS adjusting for other important factors. RESULTS: Among 388 eligible patients, 174 had leiomyosarcoma (LMS), 136 had undifferentiated pleomorphic sarcoma (UPS), and 78 had liposarcoma. OS for male versus female patients appeared to be slightly better among the full cohort [HR = 0.89; 95% confidence interval (CI), 0.66-1.20]; this association appeared to be stronger among the subsets of patients with LMS (HR = 0.76; 95% CI, 0.39-1.49) or liposarcoma (HR = 0.74; 95% CI, 0.32-1.70). Better OS for male versus female patients was also observed among all molecular subgroups except mutRB1 and mutATRX, especially among patients whose tumor retained wtTP53 (HR = 0.73; 95% CI, 0.44-1.18), wtCDKN2A (HR = 0.85; 95% CI, 0.59-1.23), wtRB1 (HR = 0.73; 95% CI, 0.51-1.04), and among patients whose tumor had mutPTEN (HR = 0.37; 95% CI, 0.09-1.62). OS also appeared to be better for males in the MSK-IMPACT and TCGA datasets. CONCLUSIONS: A fairly consistent pattern of apparent better OS for males across histologic and molecular subgroups of STS was observed. If confirmed, our results could have implications for clinical practice for prognostic stratification and possibly treatment tailoring as well as for future clinical trials design.

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.

Sex- and Co-Mutation-Dependent Prognosis in Patients with SMARCA4-Mutated Malignancies.

BACKGROUND: Whether sex and co-mutations impact prognosis of patients with SMARCA4-mutated (mutSMARCA4) malignancies is not clear. METHODS: This cohort included patients from Northern California Kaiser Permanente with next-generation sequencing (NGS) performed from August 2020 to October 2022. We used Cox regression modeling to examine the association between sex and overall survival (OS), adjusting for demographics, performance status, Charlson comorbidity index, receipt of treatment, tumor mutation burden (TMB), and TP53, KRAS, CDKN2A, STK11, and Keap1 co-mutations. RESULTS: Out of 9221 cases with NGS performed, 125 cases (1.4%) had a mutSMARCA4. The most common malignancies with a mutSMARCA4 were non-small cell lung cancer (NSCLC, 35.2%), esophageal and stomach adenocarcinoma (12.8%), and cancer of unknown primary (11.2%). The most common co-mutations were p53 (mutp53, 59.2%), KRAS (mutKRAS, 28.8%), CDKN2A (mutCDKN2A, 31.2%), STK11 (mutSTK11, 12.8%), and Keap1 (mutKeap1, 8.8%) mutations. Male patients had substantially worse OS than female patients both among the entire mutSMARCA4 cohort (HR = 1.71, [95% CI 0.92-3.18]) with a median OS of 3.0 versus 43.3 months (p < 0.001), and among the NSCLC subgroup (HR = 14.2, [95% CI 2.76-73.4]) with a median OS of 2.75 months versus un-estimable (p = 0.02). Among all patients with mutSMARCA4, mutp53 versus wtp53 (HR = 2.12, [95% CI 1.04-4.29]) and mutSTK11 versus wtSTK11 (HR = 2.59, [95% CI 0.87-7.73]) were associated with worse OS. Among the NSCLC subgroup, mutp53 versus wtp53 (HR = 0.35, [0.06-1.97]) and mutKRAS versus wtKRAS (HR = 0.04, [0.003-.45]) were associated with better OS, while mutCDKN2A versus wtCDKN2A (HR = 5.04, [1.12-22.32]), mutSTK11 versus wtSTK11 (HR = 13.10, [95% CI 1.16-148.26]), and mutKeap1 versus wtKeap1 (HR = 5.06, [95% CI 0.89-26.61}) were associated with worse OS. CONCLUSION: In our cohort of patients with mutSMARCA4, males had substantially worse prognosis than females, while mutTP53, mutKRAS, mutCDKN2A, mutSTK11 and mutKeap1were differentially associated with prognosis among all patients and among the NSCLC subgroup. Our results, if confirmed, could suggest potentially unidentified mechanisms that underly this sex and co-mutation-dependent prognostic disparity among patients whose tumor bears a mutSMARCA4.

TP53 Gain-of-Function and Non-Gain-of-Function Mutations Are Associated With Differential Prognosis in Advanced Pancreatic Ductal Adenocarcinoma.

PURPOSE: To examine the impact of TP53 gain-of-function (GOF) and non-GOF mutations on prognosis of advanced pancreatic ductal adenocarcinoma (PDAC) among patients with KRAS, CDKN2A, and SMAD4 comutations. METHODS: This cohort included patients with locally advanced, recurrent, and de novo metastatic PDAC with next-generation sequencing performed from November 2017 to May 2022. We defined R175H, R248W, R248Q, R249S, R273H, R273L, and R282W as GOF and all other p53 mutations (mutp53) as non-GOF. We used Cox regression modeling to examine the association between GOF and non-GOF mutp53 and overall survival (OS), adjusting for demographics, performance status, Charlson comorbidity index, receipt of chemotherapy, and KRAS, CDKN2A, and SMAD4 comutations. RESULTS: Of 893 total eligible patients, 68.5% had tumors with mutp53, 90.1% had KRAS mutations (mutKRAS), 44.7% had CDKN2A mutations (mutCDKN2A), and 17.0% had SMAD4 mutations. Among patients with mutp53, 121 had GOF and 491 had non-GOF. GOF mutp53 was associated with worse OS than non-GOF mutp53 (hazard ratio [HR], 1.27; 95% CI, 1.02 to 1.59) and wild-type p53 (wtp53; HR, 1.24; 95% CI, 0.98 to 1.57), whereas non-GOF was not associated with worse OS than wtp53 (HR, 0.95; 95% CI, 0.80 to 1.13). In addition, mutKRAS was associated with worse OS than wild-type KRAS in patients with mutCDKN2A (HR, 1.57; 95% CI, 0.88 to 2.80) but not in patients with wild-type CDKN2A (HR, 1.03; 95% CI, 0.76 to 1.39). CONCLUSION: GOF and non-GOF mutp53 were associated with differential prognosis in advanced PDAC. The adverse effect of mutKRAS on OS appeared to be primarily driven by patients with mutCDKN2A. Our results provide new insight that could be helpful for prognostic stratification in clinical practice and for aiding future clinical trial designs.

TP53 Gain-of-Function and Non-Gain-of-Function Mutations Are Differentially Associated With Sidedness-Dependent Prognosis in Metastatic Colorectal Cancer.

PURPOSE: To examine the association of gain-of-function (GOF) and non-gain-of-function (non-GOF) TP53 mutations with prognosis of metastatic right-sided (RCC) versus left-sided colorectal cancer (LCC). METHODS: This cohort study included patients with metastatic colorectal cancer (CRC) who had next-generation sequencing performed from November 2017 to January 2021. We defined R175H, R248W, R248Q, R249S, R273H, R273L, and R282W as GOF and all other mutp53 as non-GOF. We used Cox regression modeling to examine the association between GOF and non-GOF mutp53 and overall survival (OS), adjusting for age, sex, ethnicity, performance status, Charlson comorbidity index and receipt of chemotherapy. RESULTS: Of total 1,043 patients, 735 had tumors with mutp53 and 308 had wild-type p53 (wtp53). GOF was associated with worse OS than non-GOF mutp53 only in LCC (hazard ratio [HR] = 1.66 [95% CI, 1.20 to 2.29]), but not in RCC (HR = 0.79 [95% CI, 0.49 to 1.26]). Importantly, RCC was associated with worse OS than LCC only in the subset of patients whose CRC carried non-GOF (HR = 1.76 [95% CI, 1.30 to 2.39]), but not GOF mutp53 (HR = 0.92 [95% CI, 0.55 to 1.53]) or wtp53 (HR = 0.88 [95% CI, 0.60 to 1.28]). These associations were largely unchanged after also adjusting for RAS, BRAF, and PIK3CA mutations, and microsatellite instability-high. CONCLUSION: Poorer survival of patients with metastatic RCC versus LCC appeared to be restricted to the subset with non-GOF mutp53, whereas GOF versus non-GOF mutp53 was associated with poorer survival only among patients with LCC. This approach of collectively classifying mutp53 into GOF and non-GOF provides new insight for prognostic stratification and for understanding the mechanism of sidedness-dependent prognosis. If confirmed, future CRC clinical trials may benefit from incorporating this approach.

ASB2 is a direct target of FLI1 that sustains NF-κB pathway activation in germinal center-derived diffuse large B-cell lymphoma.

BACKGROUND: Diffuse large B-cell lymphoma (DLBCL) comprises at least two main biologically distinct entities: germinal center B-cell (GCB) and activated B-cell (ABC) subtype. Albeit sharing common lesions, GCB and ABC DLBCL present subtype-specific oncogenic pathway perturbations. ABC DLBCL is typically characterized by a constitutively active NF-kB. However, the latter is seen in also 30% of GCB DLBCL. Another recurrent lesion in DLBCL is an 11q24.3 gain, associated with the overexpression of two ETS transcription factors, ETS1 and FLI1. Here, we showed that FLI1 is more expressed in GCB than ABC DLBCL and we characterized its transcriptional network. METHODS: Gene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013 and GSE10846. ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNAs) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was done using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler. ChIP-Seq and RNA-Seq data from GCB DLBCL cell lines after FLI1 downregulation were integrated to identify putative direct targets of FLI1. RESULTS: Analysis of clinical DLBCL specimens showed that FLI1 gene was more frequently expressed at higher levels in GCB than in ABC DLBCL and its  protein levels were higher in GCB than in ABC DLBCL cell lines. Genes negatively regulated by FLI1 included tumor suppressor genes involved in negative regulation of cell cycle and hypoxia. Among positively regulated targets of FLI1, we found genes annotated for immune response, MYC targets, NF-κB and BCR signaling and NOTCH pathway genes. Of note, direct targets of FLI1 overlapped with genes regulated by ETS1, the other transcription factor gained at the 11q24.3 locus in DLBCL, suggesting a functional convergence within the ETS family. Positive targets of FLI1 included the NF-κB-associated ASB2, a putative essential gene for DLBCL cell survival. ASB2 gene downregulation was toxic in GCB DLBCL cell lines and induced NF-κB inhibition via downregulation of RelB and increased IκBα. Additionally, downregulation of FLI1, but not ASB2, caused reduction of NF-κB1 and RelA protein levels. CONCLUSIONS: We conclude that FLI1 directly regulates a network of biologically crucial genes and processes in GCB DLBCL. FLI1 regulates both the classical NF-κB pathway at the transcriptional level, and the alternative NF-κB pathway, via ASB2. FLI1 and ASB2 inhibition represents a potential novel therapeutic approach for GCB DLBCL.

Role of ETS1 in the Transcriptional Network of Diffuse Large B Cell Lymphoma of the Activated B Cell-Like Type.

Diffuse large B cell lymphoma (DLBCL) is a heterogenous disease that has been distinguished into at least two major molecular entities, the germinal center-like B cell (GCB) DLBCL and activated-like B cell (ABC) DLBCL, based on transcriptome expression profiling. A recurrent ch11q24.3 gain is observed in roughly a fourth of DLBCL cases resulting in the overexpression of two ETS transcription factor family members, ETS1 and FLI1. Here, we knocked down ETS1 expression by siRNA and analyzed expression changes integrating them with ChIP-seq data to identify genes directly regulated by ETS1. ETS1 silencing affected expression of genes involved in B cell signaling activation, B cell differentiation, cell cycle, and immune processes. Integration of RNA-Seq (RNA sequencing) data and ChIP-Seq (chromatin immunoprecipitation sequencing) identified 97 genes as bona fide, positively regulated direct targets of ETS1 in ABC-DLBCL. Among these was the Fc receptor for IgM, FCMR (also known as FAIM3 or Toso), which showed higher expression in ABC- than GCB-DLBCL clinical specimens. These findings show that ETS1 is contributing to the lymphomagenesis in a subset of DLBCL and identifies FCMR as a novel target of ETS1, predominantly expressed in ABC-DLBCL.

The ETS Inhibitors YK-4-279 and TK-216 Are Novel Antilymphoma Agents.

PURPOSE: Transcription factors are commonly deregulated in cancer, and they have been widely considered as difficult to target due to their nonenzymatic mechanism of action. Altered expression levels of members of the ETS-transcription factors are often observed in many different tumors, including lymphomas. Here, we characterized two small molecules, YK-4-279 and its clinical derivative, TK-216, targeting ETS factors via blocking the protein-protein interaction with RNA helicases, for their antilymphoma activity. EXPERIMENTAL DESIGN: The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination; validation experiments on in vivo models; and transcriptome and coimmunoprecipitation experiments. RESULTS: YK-4-279 and TK-216 demonstrated an antitumor activity across several lymphoma cell lines, which we validated in vivo. We observed synergistic activity when YK-4-279 and TK-216 were combined with the BCL2 inhibitor venetoclax and with the immunomodulatory drug lenalidomide. YK-4-279 and TK-216 interfere with protein interactions of ETS family members SPIB, in activated B-cell-like type diffuse large B-cell lymphomas, and SPI1, in germinal center B-cell-type diffuse large B-cell lymphomas. CONCLUSIONS: The ETS inhibitor YK-4-279 and its clinical derivative TK-216 represent a new class of agents with in vitro and in vivo antitumor activity in lymphomas. Although their detailed mechanism of action needs to be fully defined, in DLBCL they might act by targeting subtype-specific essential transcription factors.

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.

Preclinical evaluation of the BET bromodomain inhibitor BAY 1238097 for the treatment of lymphoma.

The epigenome is often deregulated in cancer and treatment with inhibitors of bromodomain and extra-terminal proteins, the readers of epigenetic acetylation marks, represents a novel therapeutic approach. Here, we have characterized the anti-tumour activity of the novel bromodomain and extra-terminal (BET) inhibitor BAY 1238097 in preclinical lymphoma models. BAY 1238097 showed anti-proliferative activity in a large panel of lymphoma-derived cell lines, with a median 50% inhibitory concentration between 70 and 208 nmol/l. The compound showed strong anti-tumour efficacy in vivo as a single agent in two diffuse large B cell lymphoma models. Gene expression profiling showed BAY 1238097 targeted the NFKB/TLR/JAK/STAT signalling pathways, MYC and E2F1-regulated genes, cell cycle regulation and chromatin structure. The gene expression profiling signatures also highly overlapped with the signatures obtained with other BET Bromodomain inhibitors and partially overlapped with HDAC-inhibitors, mTOR inhibitors and demethylating agents. Notably, BAY 1238097 presented in vitro synergism with EZH2, mTOR and BTK inhibitors. In conclusion, the BET inhibitor BAY 1238097 presented promising anti-lymphoma preclinical activity in vitro and in vivo, mediated by the interference with biological processes driving the lymphoma cells. Our data also indicate the use of combination schemes targeting EZH2, mTOR and BTK alongside BET bromodomains.

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.

The transcription factor ETS1 in lymphomas: friend or foe?

ETS1 is a member of the ETS family of transcription factors, which contains many cancer genes. ETS1 gene is mapped at 11q24.3, a chromosomal region that is often the site of genomic rearrangements in hematological cancers. ETS1 is expressed in a variety of cells, including B and T lymphocytes. ETS1 is important in various biological processes such as development, differentiation, proliferation, apoptosis, migration and tissue remodeling. It acts as an oncogene controlling invasive and angiogenic behavior of malignant cells in multiple human cancers. In particular, ETS1 deregulation has been reported in diffuse large B-cell lymphoma, in Burkitt lymphoma and in Hodgkin lymphoma. Here, we summarize the function of ETS1 in normal cells, with a particular emphasis on lymphocytes, and its possible role as an oncogene or tumor suppressor gene in the different mature B cell lymphomas.

An MLL-dependent network sustains hematopoiesis.

The histone methyltransferase Mixed Lineage Leukemia (MLL) is essential to maintain hematopoietic stem cells and is a leukemia protooncogene. Although clustered homeobox genes are well-characterized targets of MLL and MLL fusion oncoproteins, the range of Mll-regulated genes in normal hematopoietic cells remains unknown. Here, we identify and characterize part of the Mll-dependent transcriptional network in hematopoietic stem cells with an integrated approach by using conditional loss-of-function models, genomewide expression analyses, chromatin immunoprecipitation, and functional rescue assays. The Mll-dependent transcriptional network extends well beyond the previously appreciated Hox targets, is comprised of many characterized regulators of self-renewal, and contains target genes that are both dependent and independent of the MLL cofactor, Menin. Interestingly, PR-domain containing 16 emerged as a target gene that is uniquely effective at partially rescuing Mll-deficient hematopoietic stem and progenitor cells. This work highlights the tissue-specific nature of regulatory networks under the control of MLL/Trithorax family members and provides insight into the distinctions between the participation of MLL in normal hematopoiesis and in leukemia.

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.