TRAF3 loss-of-function reveals the noncanonical NF-κB pathway as a therapeutic target in diffuse large B cell lymphoma.

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

Hepatitis C Virus Screening among Medicaid-Insured Individuals with Opioid Use Disorder across Substance Use Disorder Treatment Settings.

Objective: Although the rapid increase in opioid use disorders (OUD) and concurrent increase in Hepatitis C virus (HCV) in the United States is well-documented, little is known about HCV testing among high-risk populations. We examine patterns of HCV testing across OUD treatment settings for individuals with OUD in New York. Methods: Using 2014 New York Medicaid claims data, we identified OUD diagnosis, OUD treatment (methadone, buprenorphine, naltrexone, other treatment (inpatient or outpatient non-medication-based psychosocial treatment, such as psychotherapy) and no treatment) utilization and HCV-testing status among beneficiaries. We performed multivariable logistic regression to identify factors associated with HCV screening across OUD treatment settings. Results: 79,764 individuals with OUD diagnoses were identified in 2014. The prevalence of HCV screening was 32.4%, 16.2%, 20.6%, 16.8%, and 18.1% for those receiving methadone, buprenorphine, naltrexone, other treatment, and no treatment, respectively. In the adjusted logistic regression, those receiving any OUD treatment had greater odds of being screened, with the highest odds among methadone clients. Conclusions: Engagement in medication for OUD is associated with increased HCV testing. Findings indicate the importance of access to medication-based treatment for OUD and a need to further improve HCV screening rates.

Belief patterns and drug use in a sample of Brazilian youth: an exploratory latent class analysis

Objective: Adolescent substance abuse is a public health concern worldwide, and its prevention is the subject of numerous programmatic efforts. Yet, little research exists on the structure of drug-related belief patterns in youth and their utility in preventive program planning. The aim of this study is to determine the structure of drug-related beliefs among 12-15-year-old students in Brazil using latent class analysis. Methods: De-identified survey data were obtained from the baseline sample (n=6,176) of a randomized controlled trial on the #Tamojunto drug use prevention program in Brazilian middle schools. Using 11 survey items assessing drug-related beliefs as indicators, four models were run and assessed for goodness-of-fit. For the best fitting model, demographic variables and substance use across latent classes were assessed. Results: Model fit statistics indicated that the best fit was a three-class solution, comprising a large Drug-Averse Beliefs class (80.9%), a smaller Permissive Beliefs class (12.7%), and an Inconsistent Beliefs class (6.4%). Respondents in the Permissive Beliefs and Inconsistent Beliefs classes reported greater past-year drug use, were slightly older and less likely to be female than those in the Drug-Averse Beliefs class. Conclusions: These results indicate that conceptualizing drug beliefs as a categorical latent variable may be useful for informing prevention. Longitudinal studies are needed to establish temporality and assess further applicability of this construct.

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.

Predictors of Medication Utilization for Opioid Use Disorder Among Medicaid-Insured HIV Patients in New York.

BACKGROUND AND OBJECTIVES: This paper investigates the prevalence and predictors for opioid use disorder (OUD) pharmacotherapy utilization for Medicaid-insured patients with human immunodeficiency virus (HIV) in New York. METHODS: We identified patients with HIV and OUD in 2014 in the New York State Medicaid claims data (n = 5621). The claims were used to identify individual client medication for addiction treatment (MAT) utilization, demographic information, and other medical and psychiatric health conditions. The logistic regression analyses were performed to explore the potential predictors of MAT service utilization among people with HIV and OUD. RESULTS: Of 5621 identified patients with HIV and OUD, 3647 (65%) received some type of MAT. Eighty-seven percent of treated patients received methadone while 10% received buprenorphine and 3% utilized both the therapies. CONCLUSIONS AND SCIENTIFIC SIGNIFICANCE: A substantial number of patients with HIV and OUD did not receive MAT. Findings suggest that there are opportunities to improve OUD care for patients with HIV and OUD, particularly among the younger generation, blacks, individuals living outside of New York City, and among those with serious psychiatric conditions. This initial study suggests that an additional research is needed to better understand how the gap in care affects this population. (Am J Addict 2020;29:151-154).

Belief patterns and drug use in a sample of Brazilian youth: an exploratory latent class analysis.

OBJECTIVE: Adolescent substance abuse is a public health concern worldwide, and its prevention is the subject of numerous programmatic efforts. Yet, little research exists on the structure of drug-related belief patterns in youth and their utility in preventive program planning. The aim of this study is to determine the structure of drug-related beliefs among 12-15-year-old students in Brazil using latent class analysis. METHODS: De-identified survey data were obtained from the baseline sample (n=6,176) of a randomized controlled trial on the #Tamojunto drug use prevention program in Brazilian middle schools. Using 11 survey items assessing drug-related beliefs as indicators, four models were run and assessed for goodness-of-fit. For the best fitting model, demographic variables and substance use across latent classes were assessed. RESULTS: Model fit statistics indicated that the best fit was a three-class solution, comprising a large Drug-Averse Beliefs class (80.9%), a smaller Permissive Beliefs class (12.7%), and an Inconsistent Beliefs class (6.4%). Respondents in the Permissive Beliefs and Inconsistent Beliefs classes reported greater past-year drug use, were slightly older and less likely to be female than those in the Drug-Averse Beliefs class. CONCLUSIONS: These results indicate that conceptualizing drug beliefs as a categorical latent variable may be useful for informing prevention. Longitudinal studies are needed to establish temporality and assess further applicability of this construct.

Single-Cell Transcriptome Analysis Reveals Disease-Defining T-cell Subsets in the Tumor Microenvironment of Classic Hodgkin Lymphoma.

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma-specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma-associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3+ T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3+ T cells in the direct vicinity of MHC class II-deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. SIGNIFICANCE: We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell-like immunosuppressive subset of LAG3+ T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints.See related commentary by Fisher and Oh, p. 342.This article is highlighted in the In This Issue feature, p. 327.

Age related medication for addiction treatment (MAT) use for opioid use disorder among Medicaid-insured patients in New York.

BACKGROUND: Medication for addiction treatment (MAT) has received much attention in recent years for treating individuals with opioid use disorders (OUD). However, these medications have been significantly underused among particular subgroups. In this paper, we describe the age distribution of treatment episodes for substance use disorder among Medicaid beneficiaries in New York and corresponding MAT use. METHODS: Using New York Medicaid claims, we identified individuals with OUD that received treatment for substance use disorder in 2015. The type of substance use treatment is the primary outcome measure, which includes methadone, buprenorphine, naltrexone or other non-medication treatment. RESULTS: A total of 88,637 individuals were diagnosed with OUD and received treatment for substance use disorder and 56,926 individuals received some type of MAT in 2015, with 40.2% receiving methadone, 21.9% receiving buprenorphine and 2.2% receiving naltrexone while 21.9% received non-medication based treatment. Young adults (ages 18-29) were a large proportion (25%) of individuals in treatment for OUD yet were the least likely to receive MAT. Relative to young adults, 30-39 year olds (adjusted odds ratio [AOR] = 1.62, 95% CI = 1.56-1.68), 40-49 year olds (AOR = 1.90, 95% CI = 1.82-1.99), 50-59 year olds (AOR = 2.65, 95% CI = 2.52-2.78), and 60-64 year olds (AOR = 5.03, 95% CI = 4.62-5.48) were more likely to receive MAT. CONCLUSIONS: These preliminary findings highlight high numbers of young adults in treatment for OUD and low rates of MAT, which is not consistent with treatment guidelines. Significant differences exist in the type of medication prescribed across age. More attention is needed to address the treatment needs among individuals of different age, notably young adults.

Somatic IL4R mutations in primary mediastinal large B-cell lymphoma lead to constitutive JAK-STAT signaling activation.

Primary mediastinal large B-cell lymphoma (PMBCL) is a distinct subtype of diffuse large B-cell lymphoma thought to arise from thymic medullary B cells. Gene mutations underlying the molecular pathogenesis of the disease are incompletely characterized. Here, we describe novel somatic IL4R mutations in 15 of 62 primary cases of PMBCL (24.2%) and in all PMBCL-derived cell lines tested. The majority of mutations (11/21; 52%) were hotspot single nucleotide variants in exon 8, leading to an I242N amino acid change in the transmembrane domain. Functional analyses establish this mutation as gain of function leading to constitutive activation of the JAK-STAT pathway and upregulation of downstream cytokine expression profiles and B cell-specific antigens. Moreover, expression of I242N mutant IL4R in a mouse xenotransplantation model conferred growth advantage in vivo. The pattern of concurrent mutations within the JAK-STAT signaling pathway suggests additive/synergistic effects of these gene mutations contributing to lymphomagenesis. Our data establish IL4R mutations as novel driver alterations and provide a strong preclinical rationale for therapeutic targeting of JAK-STAT signaling in PMBCL.

Mitogen-induced distinct epialleles are phosphorylated at either H3S10 or H3S28, depending on H3K27 acetylation.

Stimulation of the MAPK pathway results in mitogen- and stress-activated protein kinase 1/2 (MSK1/2)-catalyzed phosphorylation of histone H3 at serine 10 or 28 and expression of immediate-early (IE) genes. In 10T1/2 mouse fibroblasts, phosphorylation of H3S10 and H3S28 occurs on different H3 molecules and in different nuclear regions. Similarly, we show that mitogen-induced H3S10 and H3S28 phosphorylation occurs in separate pools in human primary fibroblasts. High-resolution imaging studies on both cell types reveal that H3S10 and H3S28 phosphorylation events can be induced in a single cell but on different alleles, giving rise to H3S10ph and H3S28ph epialleles. Coimmunoprecipitation and inhibition studies demonstrate that CBP/p300-mediated H3K27 acetylation is required for MSK1/2 to phosphorylate S28. Although the K9ac and S10ph marks coexist on H3, S10 phosphorylation is not dependent on K9 acetylation by PCAF. We propose that random targeting of H3S10 or H3S28 results from the stochastic acetylation of H3 by CBP/p300 or PCAF, a process comparable to transcriptional bursting causing temporary allelic imbalance. In 10T1/2 cells expressing Jun, at least two of three alleles per cell were induced, a sign of high expression level. The redundant roles of H3S10ph and H3S28ph might enable rapid and efficient IE gene induction.

An RCOR1 loss-associated gene expression signature identifies a prognostically significant DLBCL subgroup.

Effective treatment of diffuse large B-cell lymphoma (DLBCL) is plagued by heterogeneous responses to standard therapy, and molecular mechanisms underlying unfavorable outcomes in lymphoma patients remain elusive. Here, we profiled 148 genomes with 91 matching transcriptomes in a DLBCL cohort treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) to uncover molecular subgroups linked to treatment failure. Systematic integration of high-resolution genotyping arrays and RNA sequencing data revealed novel deletions in RCOR1 to be associated with unfavorable progression-free survival (P = .001). Integration of expression data from the clinical samples with data from RCOR1 knockdowns in the lymphoma cell lines KM-H2 and Raji yielded an RCOR1 loss-associated gene signature comprising 233 genes. This signature identified a subgroup of patients with unfavorable overall survival (P = .023). The prognostic significance of the 233-gene signature for overall survival was reproduced in an independent cohort comprising 195 R-CHOP-treated patients (P = .039). Additionally, we discovered that within the International Prognostic Index low-risk group, the gene signature provides additional prognostic value that was independent of the cell-of-origin phenotype. We present a novel and reproducible molecular subgroup of DLBCL that impacts risk-stratification of R-CHOP-treated DLBCL patients and reveals a possible new avenue for therapeutic intervention strategies.

RNA-dependent dynamic histone acetylation regulates MCL1 alternative splicing.

Histone deacetylases (HDACs) and lysine acetyltransferases (KATs) catalyze dynamic histone acetylation at regulatory and coding regions of transcribed genes. Highly phosphorylated HDAC2 is recruited within corepressor complexes to regulatory regions, while the nonphosphorylated form is associated with the gene body. In this study, we characterized the nonphosphorylated HDAC2 complexes recruited to the transcribed gene body and explored the function of HDAC-complex-mediated dynamic histone acetylation. HDAC1 and 2 were coimmunoprecipitated with several splicing factors, including serine/arginine-rich splicing factor 1 (SRSF1) which has roles in alternative splicing. The co-chromatin immunoprecipitation of HDAC1/2 and SRSF1 to the gene body was RNA-dependent. Inhibition of HDAC activity and knockdown of HDAC1, HDAC2 or SRSF1 showed that these proteins were involved in alternative splicing of MCL1. HDAC1/2 and KAT2B were associated with nascent pre-mRNA in general and with MCL1 pre-mRNA specifically. Inhibition of HDAC activity increased the occupancy of KAT2B and acetylation of H3 and H4 of the H3K4 methylated alternative MCL1 exon 2 nucleosome. Thus, nonphosphorylated HDAC1/2 is recruited to pre-mRNA by splicing factors to act at the RNA level with KAT2B and other KATs to catalyze dynamic histone acetylation of the MCL1 alternative exon and alter the splicing of MCL1 pre-mRNA.

Mitogen- and stress-activated protein kinases 1 and 2 are required for maximal trefoil factor 1 induction.

Mitogen- and stress-activated protein kinases 1 and 2 (MSK1 and MSK2), activated downstream of the ERK- and p38-mitogen-activated protein kinase pathways are involved in cell survival, proliferation and differentiation. Following mitogenic or stress stimuli, they mediate the nucleosomal response, which includes phosphorylation of histone H3 at serine 10 (H3S10ph) coupled with transcriptional activation of immediate-early genes. While MSK1 and MSK2 are closely related, their relative roles may vary with cellular context and/or stimuli. However, our knowledge of MSK2 recruitment to immediate-early genes is limited, as research has primarily focused on MSK1. Here, we demonstrate that both MSK1 and MSK2, regulate the phorbol ester 12-O-tetradecanoylphorbol-13-acetate induced expression of the breast cancer marker gene, trefoil factor 1 (TFF1), by phosphorylating H3S10 at its 5' regulatory regions. The MSK-mediated phosphorylation of H3S10 promotes the recruitment of 14-3-3 isoforms and BRG1, the ATPase subunit of the BAF/PBAF remodeling complex, to the enhancer and upstream promoter elements of TFF1. The recruited chromatin remodeling activity leads to the RNA polymerase II carboxy-terminal domain phosphorylation at the TFF1 promoter, initiating TFF1 expression in MCF-7 breast cancer cells. Moreover, we show that MSK1 or MSK2 is recruited to TFF1 regulatory regions, but as components of different multiprotein complexes.

Immediate early response genes and cell transformation.

Cancer has been described as not one disease, but several, each with unique characteristics, symptoms, prognostics and outcomes. Underlying this complexity is a differential expression of genes, leading to a motley of phenotypes which orchestrate the hallmarks of cancer. The idea of treating, suppressing or even preventing all forms of cancer with a single form of therapy seems untenable given the complexities of these gene expression profiles. However, recent advances in the study of immediate early genes, a family of genes that are rapidly and transiently upregulated following an external stimulus such as growth factors, hormones or stress, and their ubiquitous involvement in regulating oncogenomic responses may lend itself to new and unique therapies. At the very least, understanding and targeting immediate early gene expression and function remains an untapped area in cancer prevention research, and could very well provide new resources in cancer treatment and new perspectives in directed cancer suppression. In this review, we will discuss the critical role immediate early genes play in cancer progression, and provide specific examples of immediate early gene function and inhibition.

Histone H3 phosphorylation, immediate-early gene expression, and the nucleosomal response: a historical perspective.

Histone H3 is modified at serines 10 and 28 in interphase cells following activation of the RAS-MAPK or p38-MAPK pathways by growth factors or stress. These modifications are involved in the regulation of immediate-early genes, including Jun and Fos, whose increased expression is a trademark of various cancers. This review outlines the series of discoveries that led to the characterization of these modifications, the kinase, MSK1/2, which is activated by both MAPK pathways and directs phosphorylation of H3, and the mechanistic function of these modifications in transcriptional activation. Research examining the effect of deregulated MSK1/2 in human disorders, namely cancer, is evaluated. Recently, a number of reports proposed novel, intervening pathways leading to enrichment of phosphorylated serine 10 and 28 and the activation of MSK1/2. These novel pathways predict an even more complicated signalling mechanism for cell growth, apoptosis, and the immune response, suggesting that MSK1/2 is intrinsically responsible for an even greater number of biological processes. This review proposes that MSK1/2 is an optimal target for cancer therapy, based on its fundamental role in transmitting external signals into varied responses involved in cancer development.

Genome wide study of NF-Y type CCAAT boxes in unidirectional and bidirectional promoters in human and mouse.

A subset of CCAAT boxes is known as binding sites for the transcription factor NF-Y. We characterize their number, mismatches to the consensus sequence, and locations in bidirectional and unidirectional promoter sequences in human and mouse. We confront the findings with an analytical null model of DNA sequences and find that NF-Y type CCAAT boxes play key, but distinct roles in the two types of promoters. They are found above chance in both, but in unidirectional only when having few mismatches. In bidirectional, the relative positions of multiple boxes differ from what is expected by chance, suggesting the need for contiguity. In agreement, when there are four boxes (four-box configurations), these have much lower number of mismatches than expected in bidirectional promoters alone. Positioning of the first box differs in the two types of promoters and the null model, and mismatches and positioning are found to be correlated. Finally, four-box configurations are conserved between human and mouse, supporting the relevance of the findings. We conclude that bidirectional and unidirectional promoters, while sharing some similarities, appear to possess distinct regulatory mechanisms at the sequence level.

Cell-to-cell diversity in protein levels of a gene driven by a tetracycline inducible promoter.

BACKGROUND: Gene expression in Escherichia coli is regulated by several mechanisms. We measured in single cells the expression level of a single copy gene coding for green fluorescent protein (GFP), integrated into the genome and driven by a tetracycline inducible promoter, for varying induction strengths. Also, we measured the transcriptional activity of a tetracycline inducible promoter controlling the transcription of a RNA with 96 binding sites for MS2-GFP. RESULTS: The distribution of GFP levels in single cells is found to change significantly as induction reaches high levels, causing the Fano factor of the cells' protein levels to increase with mean level, beyond what would be expected from a Poisson-like process of RNA transcription. In agreement, the Fano factor of the cells' number of RNA molecules target for MS2-GFP follows a similar trend. The results provide evidence that the dynamics of the promoter complex formation, namely, the variability in its duration from one transcription event to the next, explains the change in the distribution of expression levels in the cell population with induction strength. CONCLUSIONS: The results suggest that the open complex formation of the tetracycline inducible promoter, in the regime of strong induction, affects significantly the dynamics of RNA production due to the variability of its duration from one event to the next.

Gene expression regulation through 14-3-3 interactions with histones and HDACs.

14-3-3s are phosphoserine- or phosphothreonine-binding proteins, which are involved in a variety of cellular processes, including gene regulation, differentiation, cell cycle progression, and metabolism. Their mechanism of regulation is typically to induce a conformational change on the target phosphoprotein, which can alter its catalytic activity, localization within the cell, or interactions with other proteins. In addition, 14-3-3s can act as a scaffolding protein, leading to multiprotein complex formation on the target phosphoprotein. As such, 14-3-3s are integrated into a number of important phosphorylation-dependent signaling pathways. In this review, we will examine the interaction of 14-3-3 with chromatin and chromatin modifying enzymes, specifically with phosphorylated histone H3 and histone deacetyltransferases, respectively. Through diverse mechanisms, these interactions directly affect the expression of target genes, many of which are known oncogenes and lead to tumorigenesis and metastasis. Various research studies have indicated that the mechanism of 14-3-3 interaction and its role in transcription is complex and diverse, and likely involving additional components as yet undefined.

Role of MSK1 in the malignant phenotype of Ras-transformed mouse fibroblasts.

Activated by the RAS-MAPK signaling pathway, MSK1 is recruited to immediate-early gene (IEG) regulatory regions, where it phosphorylates histone H3 at Ser-10 or Ser-28. Chromatin remodelers and modifiers are then recruited by 14-3-3 proteins, readers of phosphoserine marks, leading to the occupancy of IEG promoters by the initiation-engaged form of RNA polymerase II and the onset of transcription. In this study, we show that this mechanism of IEG induction, initially elucidated in parental 10T1/2 murine fibroblast cells, applies to metastatic Hras1-transformed Ciras-3 cells. As the RAS-MAPK pathway is constitutively activated in Ciras-3 cells, MSK1 activity and phosphorylated H3 steady-state levels are elevated. We found that steady-state levels of the IEG products AP-1 and COX-2 were also elevated in Ciras-3 cells. When MSK1 activity was inhibited or MSK1 expression was knocked down in Ciras-3 cells, the induction of IEG expression and the steady-state levels of COX-2, FRA-1, and JUN were greatly reduced. Furthermore, MSK1 knockdown Ciras-3 cells lost their malignant phenotype, as reflected by the absence of anchorage-independent growth.