Parental leave during pediatric fellowship training: A national survey.

BACKGROUND: Until recently, no uniform requirements for parental leave (PL) existed in graduate medical education. We implemented a national survey, with the objective of ascertaining fellows' perceptions of PL policies and their impact. This is the first study to focus exclusively on pediatric subspecialty fellows. METHODS: An online survey instrument was created targeting pediatric fellows. RESULTS: The survey was accessed by 1003 (25%) of the estimated 4078 pediatric subspecialty fellows and 853 (21%) submitted surveys. Respondent demographic data paralleled the data reported by the American Board of Pediatrics. Half of respondents did not know whether their program had a written PL policy. Over 40% reported ≥ 5 weeks of paid PL. Most indicated that fellows use vacation, sick leave, and unpaid time for PL. Almost half of respondents (45%) indicated that their program's PL policy increases the stress of having a child. Fellows chose establishing/extending paid leave and intentionally fostering a more supportive program culture as the most crucial candidate improvements. The importance of equitable PL polices between parent fellows and co-fellows was an important theme of our qualitative data. Fellows feel there is a moral misalignment between the field of pediatrics' dedication to maternal and child health and current PL policies governing pediatric trainees. CONCLUSIONS: PL policies vary widely among pediatric fellowship programs and are often not known by fellows. Fellows are not satisfied with PL policies, which often exacerbate stress for new parents and burden their co-fellows. Targeted modification of several aspects of PL policies may improve their acceptance.

Circulating Plasma Tumor DNA Is Superior to Plasma Tumor RNA Detection in Ewing Sarcoma Patients: ptDNA and ptRNA in Ewing Sarcoma.

The detection of tumor-specific nucleic acids from blood increasingly is being used as a method of liquid biopsy and minimal residual disease detection. However, achieving high sensitivity and high specificity remains a challenge. Here, we perform a direct comparison of two droplet digital PCR (ddPCR)-based detection methods, circulating plasma tumor RNA and circulating plasma tumor DNA (ptDNA), in blood samples from newly diagnosed Ewing sarcoma patients. First, we developed three specific ddPCR-based assays to detect EWS-FLI1 or EWS-ERG fusion transcripts, which naturally showed superior sensitivity to DNA detection on in vitro control samples. Next, we identified the patient-specific EWS-FLI1 or EWS-ERG breakpoint from five patient tumor samples and designed ddPCR-based, patient-specific ptDNA assays for each patient. These patient-specific assays show that although plasma tumor RNA can be detected in select newly diagnosed patients, positive results are low and statistically unreliable compared with ptDNA assays, which reproducibly detect robust positive results across most patients. Furthermore, the unique disease biology of Ewing sarcoma enabled us to show that most cell-free RNA is not tumor-derived, although cell-free-DNA burden is affected strongly by tumor-derived DNA burden. Here, we conclude that, even with optimized highly sensitive and specific assays, tumor DNA detection is superior to RNA detection in Ewing sarcoma patients.

ddPCR Analysis Reveals BRAF V600E Mutations Are Infrequent in Isolated Pituitary Langerhans Cell Histiocytosis Patients.

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia with a highly variable clinical presentation affecting people of all ages. Mutations in BRAF V600E are the most identifiable molecular alteration in LCH although its incidence in pediatric patients with isolated pituitary stalk involvement is not well described. Pediatric patients with LCH and isolated pituitary stalk involvement typically present with central diabetes insipidus. Diagnosis requires a transcranial biopsy which often yields scant tissue. We sought to determine the prevalence of BRAF V600E mutations in patients with isolated pituitary stalk LCH using digital droplet polymerase chain reaction because this method requires minimal tumor DNA. We identified 8 patients with isolated pituitary stalk thickening who underwent a biopsy at Children's Hospital Colorado from January 2001 to December 2019, as well as 6 patients with systemic LCH diagnosed by biopsy in the same period as a comparison. Only one out of the 8 patients with isolated thickened pituitary stalk was found to have a detectable BRAF V600E mutation. Five out of the 6 patients with systemic LCH had a detectable BRAF V600E mutation. In our series, BRAF V600E mutations are rare in pediatric patients with LCH and isolated pituitary stalk involvement.

Pediatric ovarian Sertoli-Leydig cell tumors with heterologous rhabdomyosarcoma elements: Clinical case series and review of the literature.

Sertoli-Leydig cell tumors (SLCTs) are rare ovarian neoplasms in pediatric patients. More exceedingly rare are SLCTs that also contain heterologous rhabdomyosarcoma (RMS) elements. For these patients, there is no standardized treatment. We report four cases of pediatric SLCT with heterologous RMS elements that were successfully treated with surgical resection and adjuvant chemotherapy. All four patients are alive and remain in remission.

Emapalumab in Children with Primary Hemophagocytic Lymphohistiocytosis.

BACKGROUND: Primary hemophagocytic lymphohistiocytosis is a rare syndrome characterized by immune dysregulation and hyperinflammation. It typically manifests in infancy and is associated with high mortality. METHODS: We investigated the efficacy and safety of emapalumab (a human anti-interferon-γ antibody), administered with dexamethasone, in an open-label, single-group, phase 2-3 study involving patients who had received conventional therapy before enrollment (previously treated patients) and previously untreated patients who were 18 years of age or younger and had primary hemophagocytic lymphohistiocytosis. The patients could enter a long-term follow-up study until 1 year after allogeneic hematopoietic stem-cell transplantation or until 1 year after the last dose of emapalumab, if transplantation was not performed. The planned 8-week treatment period could be shortened or extended if needed according to the timing of transplantation. The primary efficacy end point was the overall response, which was assessed in the previously treated patients according to objective clinical and laboratory criteria. RESULTS: At the cutoff date of July 20, 2017, a total of 34 patients (27 previously treated patients and 7 previously untreated patients) had received emapalumab; 26 patients completed the study. A total of 63% of the previously treated patients and 65% of the patients who received an emapalumab infusion had a response; these percentages were significantly higher than the prespecified null hypothesis of 40% (P = 0.02 and P = 0.005, respectively). In the previously treated group, 70% of the patients were able to proceed to transplantation, as were 65% of the patients who received emapalumab. At the last observation, 74% of the previously treated patients and 71% of the patients who received emapalumab were alive. Emapalumab was not associated with any organ toxicity. Severe infections developed in 10 patients during emapalumab treatment. Emapalumab was discontinued in 1 patient because of disseminated histoplasmosis. CONCLUSIONS: Emapalumab was an efficacious targeted therapy for patients with primary hemophagocytic lymphohistiocytosis. (Funded by NovImmune and the European Commission; NI-0501-04 and NI-0501-05 ClinicalTrials.gov numbers, NCT01818492 and NCT02069899.).

Central Nervous System Involvement of Rhabdomyosarcoma: A Single Institution Experience.

The incidence of central nervous system (CNS) involvement in patients with rhabdomyosarcoma (RMS) is low, and the outcome is dismal. We present a single institution analysis of CNS involvement of pediatric RMS. In 59 patients, the prevalence of CNS involvement was 11.9% (7 patients), higher than prior reports. Of the 6 deaths from disease, all had rapid progression, with a median survival of 14 days. The higher incidence could be secondary to treatment modifications or more sensitive detection. These findings are useful for decisions at the time of CNS involvement and could lead to modifications for future RMS clinical trials.

Timing of Radiation Therapy in Pediatric Wilms Tumor: A Report From the National Cancer Database.

PURPOSE: To determine, using the National Cancer Database (NCDB), the impact of the surgery to radiation therapy interval (SRI) on survival in contemporary patients with Wilms tumor (WT). METHODS AND MATERIALS: The NCDB was queried for patients aged ≤25 years diagnosed from 2004 to 2013 with unilateral WT who underwent definitive surgery and radiation therapy. The SRI was calculated for each patient. A stratified analysis was performed based on presence of metastasis using logistic regression to calculate risk factors for prolonged SRI, with a focus on the recommended SRI according to recent Children's Oncology Group trials (by day 14) and National Wilms Tumor Study-5 (by day 9). Cox regression was performed to assess the association of SRI with overall survival. RESULTS: A total of 1488 patients were included; 32.1% had metastasis at diagnosis. Among both metastatic and nonmetastatic groups, older patients were more likely to have prolonged SRI. For those without metastasis, SRI > 14 days was associated with increased risk of mortality (hazard ratio 2.13, P = .013). Analyzing SRI as a continuous variable also demonstrated an increased risk of death with longer SRI (hazard ratio 1.04 per day, P = .006) in this group. In contrast, among patients with metastasis, no significant association between SRI and mortality was found. CONCLUSION: Early initiation of radiation therapy remains a critical component of multimodal treatment for patients with nonmetastatic WT. For nonmetastatic patients, SRI ≤ 14 days correlates with improved overall survival. However, no such association was noted for patients with metastases. These results may inform the development of future WT trials.

Patterns of care and survival outcomes for adolescent and young adult patients with testicular seminoma in the United States: A National Cancer Database analysis.

INTRODUCTION: Testicular germ cell tumors (GCTs) are the most common solid tumor among adolescent and young adult (AYA) males. AYA patients with GCTs most typically have non-seminoma compared with seminoma, and accordingly there are fewer data reported on the AYA experience with testicular seminoma. OBJECTIVE: To evaluate national trends in postoperative treatment and overall survival (OS) outcomes in testicular seminoma by age group, specifically comparing AYAs with older adults. STUDY DESIGN: The National Cancer Data Base (NCDB) was queried for patients with testicular seminoma diagnosed between 2004 and 2012, who underwent orchiectomy followed by observation or adjuvant therapy (chemotherapy, radiation (RT), or both). Patients were grouped by age: AYA (15-39 years), adults between 40 and 55 years, and adults >55 years. Overall survival (OS) was presented using Kaplan-Meier curves and groups compared via a log-rank test. Univariate (UVA) and multivariate (MVA) analyses were performed using Cox proportional hazards regression models. Binary multiple logistic regression identified differences in variables by age category. RESULTS: Of the total 22,361 patients the majority were AYAs (12,880, 57.6%), followed by adults 40-55 years (8,022, 35.9%), and >55 years (1,459, 6.5%). Unadjusted 5-year OS was significantly better for AYAs versus adults 40-55 years and >55 years (98.0%, 96.4%, 87.7%; p < 0.001), as was 10-year OS (96.1%, 91.8%, 71.3% respectively; p < 0.001). The Table shows that on a MVA, OS was significantly better for AYAs versus adults 40-55 years and adults >55 years. AYA patients were also more commonly treated at centers with greater clinical volume. Additionally, AYA patients were less likely to present with metastatic disease. Accordingly, AYA patients were less likely to undergo retroperitoneal lymph node dissection (OR 0.81; p = 0.001) and were less often managed with adjuvant therapy including chemotherapy (OR 0.91; p = 0.027), RT (OR 0.93; p = 0.025), or both (OR 0.68; p = 0.020). DISCUSSION: AYA patients with testicular seminoma present with earlier stage disease and in the clinical Stage I setting are more often are managed with active surveillance following orchiectomy when compared with older adults in this population-based analysis. Among AYA patients, OS was modestly better when compared with adults 40-55 years and significantly better when compared with adults >55 years. CONCLUSION: Our objective to describe the patterns of care and survival outcomes for AYA patients with testicular seminoma in the USA was met by reviewing this large national dataset. These results may inform future guidelines for management of AYA seminoma.

Reliability of intraoperative frozen section for the diagnosis of renal tumors suspicious for malignancy in children and adolescents.

BACKGROUND: The ability of intraoperative frozen section (IFS) to reliably diagnose renal tumors in children and adolescents is largely unknown. The objective of our study is to evaluate the ability of IFS to establish a histologic diagnosis for renal tumors in this population. METHODS: We reviewed our experience with patients who underwent IFS at the time of surgery for a renal tumor suspicious for malignancy from 2005 to 2015. The IFS was compared to the final pathology (FP). Data on concordance and reliability were analyzed. RESULTS: One hundred thirty patients underwent surgical interventions for a renal tumor suspicious for malignancy, and 32 (25%) patients underwent IFS. Median turnaround time for IFS was 20 min (range 13-44). The histologic IFS diagnosis correlated with FP in 26 (81.2%) cases was discrepant in three (9.4%) cases, and IFS was deferred to FP in three (9.4%) cases (kappa 0.71, 95% confidence interval [CI]: 0.52-0.899, P < 0.001). The IFS correctly distinguished between Wilms tumor and non-Wilms tumor in 30 (94%) cases (kappa 0.874, 95% CI: 0.705-1, P < 0.001). A total of 17 of 19 (89.5%) Wilms tumors were correctly diagnosed by IFS, yielding a sensitivity of 0.89 (95% CI: 0.67-0.99) and a specificity of 1 (95% CI: 0.75-1). CONCLUSION: IFS is a reliable tool to establish a histologic diagnosis and to differentiate between Wilms and non-Wilms tumors in children and adolescents with renal tumors. The use of IFS should be encouraged in cases in which obtaining a diagnosis will provide guidance for important "real-time" medical decision making, specifically additional adjunctive surgical procedures.

Survival outcomes of adolescent and adult patients with non-seminomatous testicular germ-cell tumors: A population-based study.

BACKGROUND: In adolescents, approximately 90% of testicular germ cell tumors (T-GCTs) are non-seminomas (NS T-GCTs). Few studies have evaluated the impact of age, specifically in adolescence, on outcomes of NS T-GCTs. OBJECTIVE: The purpose of this study was to review all patients diagnosed with NS T-GCTs in the Surveillance, Epidemiology, and End Results (SEER) database to evaluate the association between age (adolescents vs. adults) and survival outcomes. METHOD: The SEER database was queried for individuals ≥13 years old diagnosed with NS T-GCTs from 1995 to 2012. Patients were categorized into adolescent (13-19 years) and adult (≥20 years) cohorts. A Cox proportional hazards model was used for multivariate analysis (MVA). RESULTS: A total of 13,963 patients (1496 adolescents, 12,467 adults) was included. Median follow-up was 71 months (range 1-215). Five-year overall survival (OS) for adolescent and adult patients was 94% and 92%, respectively (p = 0.007); 5-year cancer-specific survival (CSS) was 95% and 94%, respectively (p = 0.139). Under MVA, adolescent patients had improved OS (HR 0.61; 95% CI 0.50-0.75; p < 0.001) and CSS (HR 0.65; 95% CI 0.51-0.82; p < 0.001), when compared with adults (Table). In a logistic regression analysis adjusting for demographics, adolescent patients were more likely to present with regional or distant metastatic disease (OR 1.16; 95% CI 1.01-1.35; p = 0.039), undergo an orchiectomy (OR 2.44; 95% CI 1.50-4.00; p < 0.001) or tumor excision (OR 2.43; 95% CI 1.57-3.77; p < 0.001), and receive other adjuvant surgery (OR 5.87; 95% CI 2.25-15.30; p < 0.001). CONCLUSIONS: To our knowledge, this is the largest population-based comparative analysis in NS T-GCTs comparing outcomes between these two age groups. Adolescent patients with NS T-GCTs had slightly improved survival compared with adults, despite presenting with more advanced disease. While adolescent patients present at more advanced stage, they achieve excellent survival outcomes possibly at the cost of a greater therapeutic burden.

Cardiac Mortality in Children and Adolescents with Hodgkin's Lymphoma: A Surveillance, Epidemiology and End Results Analysis.

PURPOSE: The purpose of this study was to evaluate the risk of cardiac death in pediatric Hodgkin's lymphoma (HL) survivors and identify high-risk groups that may need additional surveillance. METHODS: The Surveillance, Epidemiology and End Results program database was queried to analyze the rates of radiation therapy (RT) use and cardiac-specific mortality (CSM) in HL patients, aged 0-21 years, treated from 1973 to 2007. Primary endpoint was cardiac mortality. RESULTS: A total of 6552 patients were included. Median follow-up was 12 years (range, 0-40). Median age at diagnosis was 17 years (range, 0-21). The majority were white (85.5%), from western states (41.2%), had nodular sclerosis HL (73.2%), presented with stage I or II disease (51.5%), and received RT (56.1%). Death from cardiac disease occurred in 114 patients (9.2% of all deaths). CSM for the entire cohort at 10-, 20-, and 30-year time points was 0.3%, 1.6%, and 5.0%, respectively. Median age at the time of cardiac death was 39 years (range, 18-58 years). Under multivariate analysis (MVA), adolescent patients (ages 13-21) had higher rates of CSM (hazard ratio [HR], 3.05; p = 0.005). Female gender (HR, 0.43; p < 0.001), patients treated from 1998 to 2007 (HR, 0.19; p = 0.018), and those with lymphocyte-rich histology (HR, 0.14; p = 0.047) had significantly lower rates of CSM. Use of RT was not associated with CSM under MVA (HR, 1.18, p = 0.452). CONCLUSION: The cumulative incidence of CSM in this population analysis of pediatric HL was 9.2%, with a steady decline over the past several decades. Adolescent patients at diagnosis and males were more likely to die of cardiac-related causes.

A multidisciplinary approach to the management of anterior mediastinal masses in children.

PURPOSE: Anterior mediastinal masses (AMM) pose a diagnostic challenge to surgeons, oncologists, anesthesiologists, intensivists, and interventional radiologists as induction of general anesthesia can cause airway obstruction and cardiovascular collapse. We hypothesized that in the majority of patients, diagnosis can be obtained through biopsy of extrathoracic tissue. METHODS: We performed a retrospective review of all patients in the solid tumor oncology clinic with a diagnosis of AMM between 2002 and 2012 including preoperative evaluation and management prior to obtaining a tissue diagnosis, clinical course and complications. RESULTS: We identified 69 patients with AMM (mean age 12.2±4.4years, 64% male) secondary to Hodgkin lymphoma (34), Non-Hodgkin lymphoma (26), and other diagnoses (9). The majority of patients (56, 81.2%) underwent biopsy of tissue outside of the mediastinal mass. Local anesthesia with sedation was used for successful biopsy in 21 (30%) patients. Four (5.8%) required repeat biopsy due to inadequate sample obtained at initial procedure. Three (4.4%) suffered respiratory complications with no fatalities or severe complications. CONCLUSIONS: Our data demonstrate that in the majority of children with AMM, tissue biopsy can be successfully obtained from tissue outside of the mass itself with minimal complications and highlight the importance of multidisciplinary preoperative planning to minimize anesthetic risks.

Minimally invasive surgical techniques are safe in the diagnosis and treatment of pediatric malignancies.

BACKGROUND: Minimally invasive surgical (MIS) techniques have become an established part of the care of the adult oncology patient. As surgeons have become more experienced with these advances in technique, MIS has recently seen an expanding role in the diagnosis and treatment of pediatric malignancies. We hypothesize that MIS techniques can be used to provide reliable diagnosis and safe therapeutic resection of many pediatric malignancies. PROCEDURE: We performed a retrospective review of all patients who underwent a minimally invasive operation for diagnosis or treatment of a malignant solid tumor at the Children's Hospital Colorado over a ten-year period. RESULTS: A total of 105 minimally invasive procedures were performed in 98 patients, 61% of which were male. The majority of cases, 78 (74%) were thoracoscopic procedures and the remaining 27 (26%) were laparoscopic procedures. Twenty-one (27%) thoracoscopic procedures were performed for complete resection of primary tumor or metastases, with only three cases (14%) converted to open thoracotomy. Tumors that were successfully removed thoracoscopically include neuroblastomas (n = 8), metastatic disease (n = 7), and a schwannoma. Of the 28 laparoscopic procedures, nine were performed for tumor resection with one case converted to open. Tumors that were successfully removed laparoscopically include 6 adrenal neuroblastomas and one pseudopapillary pancreatic tumor. There were no major surgical complications. No port site or surgical site recurrences were reported. CONCLUSIONS: MIS techniques can be used safely and effectively for the diagnosis and resection of pediatric malignancies and treatment decisions can be made accurately based on tissue obtained.

MAP kinase kinase kinase-2 (MEKK2) regulates hypertrophic remodeling of the right ventricle in hypoxia-induced pulmonary hypertension.

Pulmonary hypertension (PH) results in pressure overload of the right ventricle (RV) of the heart, initiating pathological RV remodeling and ultimately leading to right heart failure. Substantial research indicates that signaling through the MAPK superfamily mediates pathological cardiac remodeling. These considerations led us to test the hypothesis that the regulatory protein MAPKKK-2 (MEKK2) contributes to RV hypertrophy in hypoxia-induced PH. Transgenic mice with global knockout of MEKK2 (MEKK2(-/-) mice) and age-matched wild-type (WT) mice were exposed to chronic hypobaric hypoxia (10% O(2), 6 wk) and compared with animals under normoxia. Exposure to chronic hypoxia induced PH in WT and MEKK2(-/-) mice. In response to PH, WT mice showed RV hypertrophy, demonstrated as increased ratio of RV weight to body weight, increased RV wall thickness at diastole, and increased cardiac myocyte size compared with normoxic control animals. In contrast, each of these measures of RV hypertrophy seen in WT mice after chronic hypoxia was attenuated in MEKK2(-/-) mice. Furthermore, chronic hypoxia elicited altered programs of hypertrophic and inflammatory gene expression consistent with pathological RV remodeling in WT mice; MEKK2 deletion selectively inhibited inflammatory gene expression compared with WT mice. The actions of MEKK2 were mediated in part through regulation of the abundance and phosphorylation of its effector, ERK5. In conclusion, signaling by MEKK2 contributes to RV hypertrophy and altered myocardial inflammatory gene expression in response to hypoxia-induced PH. Therapies targeting MEKK2 may protect the myocardium from hypertrophy and pathological remodeling in human PH.

Pediatric developmental therapies: interesting new drugs now in early-stage clinical trials.

The current high cure rates for children diagnosed with cancer can be attributed in part to emphasis on large cooperative group clinical trials. The significant improvement in pediatric cancer survival over the past few decades is the result of optimized chemotherapy drug dosing, timing, and intensity; however, further alterations in traditional chemotherapy agents are unlikely to produce substantially better outcomes. Furthermore, there remains a subset of patients who have a very poor prognosis due to tumor type or stage at presentation, or who have a dismal prognosis with relapse or recurrence. As such, innovative approaches to therapy and new drugs are clearly needed for introduction into the current pediatric oncology arsenal. A variety of biologically targeted therapies that have shown promise in preclinical studies and early-phase adult clinical trials are now being explored in pediatric clinical trials. These novel agents hold the promise for continuing to drive forward improvements in patient survival, with potentially less toxicity than exists with traditional chemotherapy drugs.

Simultaneous occurrence of right adrenocortical tumor and left adrenal neuroblastoma in an infant with Beckwith-Wiedemann syndrome.

Children with Beckwith-Wiedemann syndrome (BWS) have increased risk for development of embryonal tumors. We present the case of an infant with BWS who has hypomethylation of LIT1 gene in the 11p15.5 chromosomal region and at 6 months of age presented with simultaneous occurrence of neuroblastoma arising from the left adrenal gland and a right adrenocortical tumor. She underwent surgical resection of both tumors and remains tumor free 18 months after surgery.

The new University of Colorado medical school curriculum: a pediatric perspective.

The University of Colorado School of Medicine has developed an innovative 4-year undergraduate curriculum. As a strong advocate for education and curriculum reform, Dr M. Douglas Jones Jr. created an environment for pediatrics to flourish in this new curriculum. Pediatric content has increased in all years of the curriculum, and pediatric faculty have had greater opportunities to teach and seek career development in medical education. In this report, we review the process that led to curriculum reform, provide an overview of the new curriculum design, and highlight examples of the positive impact this process has had on education in pediatrics. We hope that sharing our experience, may benefit others in medical education.

MEKK1 regulates the AP-1 dimer repertoire via control of JunB transcription and Fra-2 protein stability.

Activator protein 1 (AP-1) transcription factor dimers are composed of Jun, Fos, and ATF member proteins, but the mechanisms that determine AP-1 composition are not clearly defined and the function of specific dimers is not well understood. MEKK1 is a mitogen-activated protein kinase (MAPK) kinase kinase and an ubiquitin ligase that regulates both the extracellular signal-regulated kinase 1/2 and the c-Jun amino-terminal kinase. Herein, we demonstrate that MEKK1 regulates the AP-1 protein repertoire. Both FGF-2 and phorbol ester-inducible urokinase-type plasminogen activator (uPA) expression requires AP-1 binding to an enhancer element in the uPA promoter, and we have previously shown that FGF-2 or PMA induction of uPA expression is strongly dependent on MEKK1. JunB mRNA is significantly increased in MEKK1-/- cells, demonstrating that MEKK1 suppresses JunB mRNA expression. Upregulation of JunB expression in MEKK1-/- cells forms an inhibitory AP-1 complex that binds to the uPA promoter and inhibits uPA transcription. MEKK1 also regulates Fra-2 protein stability by inducing Fra-2 ubiquitination and degradation. MEKK1 regulates AP-1-dependent gene expression by regulating the expression, activity and degradation of component members of the AP-1 complex. Controlling the repertoire of a transcription factor complex is a newly defined function for an MAPK kinase kinase.

MEKK2 regulates the coordinate activation of ERK5 and JNK in response to FGF-2 in fibroblasts.

Mitogen-activated protein kinases (MAPKs) are regulated by MAPK kinases (MKKs), which are in turn regulated by MKK kinases (MKKKs). While a single MKKK can regulate several different MAPK family members, and several MKKKs can often activate the same MAPK, emerging evidence indicates a unique role for individual MKKKs in acting as signaling nodes to coordinately activate different subsets of MAPKs in response to specific cellular stimuli. Thus, while there is much apparent overlap in MAPK regulation by different MKKKs, each MKKK serves a specific purpose in regulation of unique cellular functions. The purpose of this study was to define the specific role of MEKK2, an MKKK, in MAPK regulation and cell function. MEKK2 coordinately activates the ERK5 and JNK pathways. Targeted disruption of MEKK2 expression causes loss of ERK5 and JNK activation in response to FGF-2 in mouse embryonic fibroblasts (MEFs). FGF-2 receptor signaling requires MEKK2 for induction of mRNA for c-Jun, Fra-1, and Fra-2, components of the AP-1 transcription complex. In FGF-2-stimulated MEKK2-/- fibroblasts, c-Jun phosphorylation is inhibited, consistent with a loss of JNK activation. Thus, MEKK2 regulates AP-1 activity at two levels, by regulating both expression of AP-1 components and c-Jun N-terminal phosphorylation. One function of the AP-1 transcription complex is to regulate cytokine gene expression. Expression of IL-1alpha, IL-1beta, IL-6, and TNFalpha is inhibited in MEKK2-/- fibroblasts. Bacterial lipopolysaccharide (LPS) and TNFalpha neither activate ERK5 nor require MEKK2 for JNK activation, demonstrating specificity of MEKK2 in FGF-2 receptor signaling and control of cytokine gene expression.

MEK kinase 2 and the adaptor protein Lad regulate extracellular signal-regulated kinase 5 activation by epidermal growth factor via Src.

Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex.