Regulation of HOX gene expression in AML.

As key developmental regulators, HOX cluster genes have varied and context-specific roles in normal and malignant hematopoiesis. A complex interaction of transcription factors, epigenetic regulators, long non-coding RNAs and chromatin structural changes orchestrate HOX expression in leukemia cells. In this review we summarize molecular mechanisms underlying HOX regulation in clinical subsets of AML, with a focus on NPM1 mutated (NPM1mut) AML comprising a third of all AML patients. While the leukemia initiating function of the NPM1 mutation is clearly dependent on HOX activity, the favorable treatment responses in these patients with upregulation of HOX cluster genes is a poorly understood paradoxical observation. Recent data confirm FOXM1 as a suppressor of HOX activity and a well-known binding partner of NPM suggesting that FOXM1 inactivation may mediate the effect of cytoplasmic NPM on HOX upregulation. Conversely the residual nuclear fraction of mutant NPM has also been recently shown to have chromatin modifying effects permissive to HOX expression. Recent identification of the menin-MLL interaction as a critical vulnerability of HOX-dependent AML has fueled the development of menin inhibitors that are clinically active in NPM1 and MLL rearranged AML despite inconsistent suppression of the HOX locus. Insights into context-specific regulation of HOX in AML may provide a solid foundation for targeting this common vulnerability across several major AML subtypes.

PRL2 Phosphatase Promotes Oncogenic KIT Signaling in Leukemia Cells through Modulating CBL Phosphorylation.

Receptor tyrosine kinase KIT is frequently activated in acute myeloid leukemia (AML). While high PRL2 (PTP4A2) expression is correlated with activation of SCF/KIT signaling in AML, the underlying mechanisms are not fully understood. We discovered that inhibition of PRL2 significantly reduces the burden of oncogenic KIT-driven leukemia and extends leukemic mice survival. PRL2 enhances oncogenic KIT signaling in leukemia cells, promoting their proliferation and survival. We found that PRL2 dephosphorylates CBL at tyrosine 371 and inhibits its activity toward KIT, leading to decreased KIT ubiquitination and enhanced AKT and ERK signaling in leukemia cells. IMPLICATIONS: Our studies uncover a novel mechanism that fine-tunes oncogenic KIT signaling in leukemia cells and will likely identify PRL2 as a novel therapeutic target in AML with KIT mutations.

Nore1 inhibits age-associated myeloid lineage skewing and clonal hematopoiesis but facilitates termination of emergency (stress) granulopoiesis.

Age-associated bone marrow changes include myeloid skewing and mutations that lead to clonal hematopoiesis. Molecular mechanisms for these events are ill defined, but decreased expression of Irf8/Icsbp (interferon regulatory factor 8/interferon consensus sequence binding protein) in aging hematopoietic stem cells may contribute. Irf8 functions as a leukemia suppressor for chronic myeloid leukemia, and young Irf8-/- mice have neutrophilia with progression to acute myeloid leukemia (AML) with aging. Irf8 is also required to terminate emergency granulopoiesis during the innate immune response, suggesting this may be the physiologic counterpart to leukemia suppression by this transcription factor. Identifying Irf8 effectors may define mediators of both events and thus contributors to age-related bone marrow disorders. In this study, we identified RASSF5 (encoding Nore1) as an Irf8 target gene and investigated the role of Nore1 in hematopoiesis. We found Irf8 activates RASSF5 transcription and increases Nore1a expression during emergency granulopoiesis. Similar to Irf8-/- mice, we found that young Rassf5-/- mice had increased neutrophils and progressed to AML with aging. We identified enhanced DNA damage, excess clonal hematopoiesis, and a distinct mutation profile in hematopoietic stem cells from aging Rassf5-/- mice compared with wildtype. We found sustained emergency granulopoiesis in Rassf5-/- mice, with repeated episodes accelerating AML, also similar to Irf8-/- mice. Identifying Nore1a downstream from Irf8 defines a pathway involved in leukemia suppression and the innate immune response and suggests a novel molecular mechanism contributing to age-related clonal myeloid disorders.

Dynamin-2 deficiency causes age- and sex-dependent neutropenia and myelodysplasia in mice.

The dynamins are a family of ubiquitously expressed GTPase proteins, best known for their role in membrane remodeling. Their contribution to hematopoiesis is incompletely recognized. Individuals with Charcot-Marie-Tooth disease with dynamin-2 (DNM2) mutations often develop neutropenia. We previously reported that dynamin (DNM) inhibition impairs SDF1a-mediated migration in megakaryocytes. Here, we report on conditionally Dnm2 deleted mice in hematopoietic tissues using the Vav-Cre murine strain. Homozygous Dnm2 deletion in blood tissues is embryonic lethal. Dnm2het male mice only developed a slightly decreased hemoglobin level. Dnm2het female mice developed leukopenia by 40 weeks of age and neutropenia by 65 weeks of age. Flow cytometry revealed decreased lineage-negative cells and granulocyte-monocyte progenitors in Dnm2het female mice. Immunohistochemical staining of bone marrow (BM) for mature neutrophils with Ly6G was decreased and myelodysplastic features were present in the BM of Dnm2het female mice. A linear distribution of Ly6G+ BM cells along blood vessels was observed in fewer Dnm2het mice than in controls, suggesting that the migration pattern in the marrow is altered. Marrow neutrophils treated with dynamin inhibitor, dynasore, showed increased cell surface CXCR4, suggesting that abnormal migration results in marrow neutrophil retention. Dnm2het female mice also developed splenomegaly secondary to germinal center hyperplasia at younger ages, suggesting perturbed immunity. In summary, female mice with BM Dnm2 haploinsufficiency developed neutropenia as they aged with decreased granulocyte progenitor production and migration defects. Our studies indicate a potential mechanism for the development of chronic idiopathic neutropenia, a disease that predominantly presents in middle-aged women.

PRL2 phosphatase enhances oncogenic FLT3 signaling via dephosphorylation of the E3 ubiquitin ligase CBL at tyrosine 371.

Acute myeloid leukemia (AML) is an aggressive blood cancer with poor prognosis. FMS-like tyrosine kinase receptor-3 (FLT3) is one of the major oncogenic receptor tyrosine kinases aberrantly activated in AML. Although protein tyrosine phosphatase PRL2 is highly expressed in some subtypes of AML compared with normal human hematopoietic stem and progenitor cells, the mechanisms by which PRL2 promotes leukemogenesis are largely unknown. We discovered that genetic and pharmacological inhibition of PRL2 significantly reduce the burden of FLT3-internal tandem duplications-driven leukemia and extend the survival of leukemic mice. Furthermore, we found that PRL2 enhances oncogenic FLT3 signaling in leukemia cells, promoting their proliferation and survival. Mechanistically, PRL2 dephosphorylates the E3 ubiquitin ligase CBL at tyrosine 371 and attenuates CBL-mediated ubiquitination and degradation of FLT3, leading to enhanced FLT3 signaling in leukemia cells. Thus, our study reveals that PRL2 enhances oncogenic FLT3 signaling in leukemia cells through dephosphorylation of CBL and will likely establish PRL2 as a novel druggable target for AML.

Assessment of a Simplified Cell-Free DNA Method for Prenatal Down Syndrome Screening.

BACKGROUND: Prenatal screening for common trisomies via cell-free (cfDNA) is usually implemented by technologies utilizing massively parallel sequencing, stringent environmental controls, complex bioinformatics, and molecular expertise. An alternative and less complex methodology utilizes rolling circle amplification (RCA). Further evaluation of its performance and related requirements are warranted. METHODS: At 16 sites, women at 10 to 20 weeks gestation provided informed consent, relevant information, and 2 to 3 blood samples. Samples shipped for testing were processed and stored. Women were enrolled at primary cfDNA screening, or following such screening at referral for diagnostic testing. RCA testing occurred post-enrollment, over 11 months. Diagnostic results and delivery notes determined clinical truth. Detection rates were based on confirmed trisomic pregnancies; false-positive rates were based on unaffected pregnancies from the general population. RESULTS: Detection rate for the common trisomies was 95.9% (117/122, 95% CI, 90.5%-98.5%); overall false-positive rate was 1.00% (22/2,205, 0.65%-1.51%). Test failure rate after repeat testing was 0.04%. When assay standard deviations were below pre-specified levels, the overall false-positive rate was much lower at 0.30% (P < 0.001). Fetal sex calls were correct for 99.7%. One technician analyzed 560 samples over 2 weeks, a rate of 14 000/year. CONCLUSIONS: Our assessment of this simplified cfDNA-based system for prenatal screening for common trisomies performed in a prenatal screening laboratory is encouraging. Improved detection, low failure rates and rapid reporting can be achieved by collecting 2 samples. Future priorities should include achieving higher run precision using a single collection tube. CLINICALTRIALS.GOV REGISTRATION NUMBER: NCT03087357.

Ruxolitinib ameliorates progressive anemia and improves survival during episodes of emergency granulopoiesis in Fanconi C-/- mice.

Fanconi anemia (FA) is an inherited disorder of DNA repair with hematologic manifestations that range from anemia to bone marrow failure to acute myeloid leukemia. In a murine model of FA (Fancc-/- mice), we found bone marrow failure was accelerated by repeated attempts to induce emergency (stress) granulopoiesis, the process for granulocyte production during the innate immune response. Fancc-/- mice exhibited an impaired granulocytosis response and died with profound anemia during repeated challenge. In the current study, we found erythropoiesis and serum erythropoietin decreased in Fancc-/- and wild-type (Wt) mice as emergency granulopoiesis peaked. Serum erythropoietin returned to baseline during steady-state resumption, and compensatory proliferation of erythroid progenitors was associated with DNA damage and apoptosis in Fancc-/- mice, but not Wt mice. The erythropoietin receptor activates Janus kinase 2 (Jak2), and we found treatment of Fancc-/- mice with ruxolitinib (Jak1/2-inhibitor) decreased anemia, enhanced granulocytosis, delayed clonal progression and prolonged survival during repeated emergency granulopoiesis episodes. This was associated with a decrease in DNA damage and apoptosis in Fancc-/- erythroid progenitors during this process. Transcriptome analysis of these cells identified enhanced activity of pathways for metabolism of reactive oxygen species, and decreased apoptosis- and autophagy-related pathways, as major ruxolitinib-effects in Fancc-/- mice. In contrast, ruxolitinib influenced primarily pathways involved in proliferation and differentiation in Wt mice. Ruxolitinib is approved for treatment of myeloproliferative disorders and graft-versus-host disease, suggesting the possibility of translational use as a bone marrow protectant in FA.

Analysis of serum HE4 levels in various histologic subtypes of epithelial ovarian cancer and other malignant tumors.

BACKGROUND: The measurement of serum HE4 levels has emerged as a sensitive and specific biomarker for epithelial ovarian cancers (EOCs). However, serum levels in women diagnosed with various histologic subtypes of EOC and in women with metastatic non-ovarian primary malignancies have not been widely reported. OBJECTIVE: The goal of this study was to identify how serum HE4 levels vary in women diagnosed with different histologic subtypes of EOC and non-ovarian malignancies. METHODS: Data from six prospective pelvic mass clinical trials was combined and an evaluation of serum HE4 levels in women diagnosed with a malignancy was performed. For all patients, serum was obtained prior to surgery and final pathology, including primary tumor site, histologic subtype, grade and stage, were recorded. The mean, median, standard deviation, maximum, and minimum HE4 levels were determined for each group. RESULTS: A total of 984 patients were included in this study, with the average patient age being 60 years old. There were 230 premenopausal and 754 postmenopausal patients. Serum HE4 levels were elevated (≥70.0 pMol) in 85%of EOCs, 40%of LMP tumors, 21%of non-EOCs (germ cell tumors), 25%of cervical cancers, and 47%of non-gynecologic metastatic cancers. Analysis of histologic subtypes revealed 90%(n = 391) of serous, 85%(n = 73) of endometrioid, 45%(n = 42) of mucinous, 86%(n = 51) of mixed tumors, and 69%(n = 36) of clear cell tumors had elevated serum HE4 levels. CONCLUSIONS: Serum HE4 levels are most often elevated in women with high grade serous and endometrioid EOCs, and though serum elevations are seen more often with advanced stage disease, HE4 is also often elevated in early stage disease and lower grade tumors.

Comparison of the Transcriptomic Signatures in Pediatric and Adult CML.

Children with chronic myeloid leukemia (CML) tend to present with higher white blood counts and larger spleens than adults with CML, suggesting that the biology of pediatric and adult CML may differ. To investigate whether pediatric and adult CML have unique molecular characteristics, we studied the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy pediatric and adult CD34+ control cells. Using high-throughput RNA sequencing, we found 567 genes (207 up- and 360 downregulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. Directly comparing pediatric and adult CML CD34+ cells, 398 genes (258 up- and 140 downregulated), including many in the Rho pathway, were differentially expressed in pediatric CML CD34+ cells. Using RT-qPCR to verify differentially expressed genes, VAV2 and ARHGAP27 were significantly upregulated in adult CML CD34+ cells compared to pediatric CML CD34+ cells. NCF1, CYBB, and S100A8 were upregulated in adult CML CD34+ cells but not in pediatric CML CD34+ cells, compared to healthy controls. In contrast, DLC1 was significantly upregulated in pediatric CML CD34+ cells but not in adult CML CD34+ cells, compared to healthy controls. These results demonstrate unique molecular characteristics of pediatric CML, such as dysregulation of the Rho pathway, which may contribute to clinical differences between pediatric and adult patients.

Inhibitory effects of Tomivosertib in acute myeloid leukemia.

The MAPK-interacting kinases 1 and 2 (MNK1/2) have generated increasing interest as therapeutic targets for acute myeloid leukemia (AML). We evaluated the therapeutic potential of the highly-selective MNK1/2 inhibitor Tomivosertib on AML cells. Tomivosertib was highly effective at blocking eIF4E phosphorylation on serine 209 in AML cells. Such inhibitory effects correlated with dose-dependent suppression of cellular viability and leukemic progenitor colony formation. Moreover, combination of Tomivosertib and Venetoclax resulted in synergistic anti-leukemic responses in AML cell lines. Mass spectrometry studies identified novel putative MNK1/2 interactors, while in parallel studies we demonstrated that MNK2 - RAPTOR - mTOR complexes are not disrupted by Tomivosertib. Overall, these findings demonstrate that Tomivosertib exhibits potent anti-leukemic properties on AML cells and support the development of clinical translational efforts involving the use of this drug, alone or in combination with other therapies for the treatment of AML.

Therapeutic Vulnerabilities of Transcription Factors in AML.

Acute myeloid leukemia (AML) is characterized by impaired myeloid lineage differentiation, uncontrolled proliferation, and inhibition of proapoptotic pathways. In spite of a relatively homogeneous clinical disease presentation, risk of long-term survival in AML varies from 20% to 80% depending on molecular disease characteristics. In recognition of the molecular heterogeneity of AML, the European Leukemia Net (ELN) and WHO classification systems now incorporate cytogenetics and increasing numbers of gene mutations into AML prognostication. Several of the genomic AML subsets are characterized by unique transcription factor alterations that are highlighted in this review. There are many mechanisms of transcriptional deregulation in leukemia. We broadly classify transcription factors based on mechanisms of transcriptional deregulation including direct involvement of transcription factors in recurrent translocations, loss-of-function mutations, and intracellular relocalization. Transcription factors, due to their pleiotropic effects, have been attractive but elusive targets. Indirect targeting approaches include inhibition of upstream kinases such as TAK1 for suppression of NFκB signaling and downstream effectors such as FGF signaling in HOXA-upregulated leukemia. Other strategies include targeting scaffolding proteins like BrD4 in the case of MYC or coactivators such as menin to suppress HOX expression; disrupting critical protein interactions in the case of ß-catenin:TCF/LEF, and preventing transcription factor binding to DNA as in the case of PU.1 or FOXM1. We comprehensively describe the mechanism of deregulation of transcription factors in genomic subsets of AML, consequent pathway addictions, and potential therapeutic strategies.

Association between a history of depression and anti-müllerian hormone among late-reproductive aged women: the Harvard study of moods and cycles.

BACKGROUND: There is conflicting evidence regarding the association between a history of depression and risk of early menopause. In a cohort of premenopausal women, we investigated the association between depression history and ovarian reserve, as measured by anti-müllerian hormone (AMH). METHODS: The Harvard Study of Moods and Cycles (HSMC) was a prospective cohort study of women living in the Boston, MA metropolitan-area (1995-1999). Women aged 36-45 years at cohort entry (1995) were sampled from seven Boston metropolitan-area communities using census directories. We measured serum AMH in early-follicular phase venous blood specimens from 141 women with a Structured Clinical Interview for DSM-IV (SCID)-confirmed history of depression and 228 without such a history. We calculated prevalence ratios (PR) for the association between characteristics of depression history and low AMH (≤1.4 ng/mL), adjusting for several potential confounders. RESULTS: The prevalence of low AMH was similar among depressed (57.5%) and non-depressed (57.9%) women (Adjusted [Adj] PR = 0.90, 95% CI: 0.75, 1.08). Among depressed women, results were not appreciably different among those who had ever used antidepressants and those with comorbid anxiety. Modest inverse associations between depression and low AMH were seen among women aged 36-40 years (Adj PR = 0.75, 95% CI: 0.52, 1.09) and nulliparous women (Adj PR = 0.77, 95% CI: 0.59, 1.00). No dose-response association with greater duration or length of depressive symptoms was observed. CONCLUSIONS: Overall, the prevalence of low AMH was similar for depressed and non-depressed women 36-45 years of age. Surprisingly, among younger and nulliparous women, those with a history of depression had a slightly reduced prevalence of low AMH relative to those without such a history. These results do not indicate reduced ovarian reserve among women with a history of depression.

An aberrantly sustained emergency granulopoiesis response accelerates postchemotherapy relapse in MLL1-rearranged acute myeloid leukemia in mice.

Acute myeloid leukemia (AML) with mixed lineage leukemia 1 (MLL1) gene rearrangement is characterized by increased expression of a set of homeodomain transcription factors, including homeobox A9 (HOXA9) and HOXA10. The target genes for these regulators include fibroblast growth factor 2 (FGF2) and Ariadne RBR E3 ubiquitin ligase 2 (ARIH2). FGF2 induces leukemia stem cell expansion in MLL1-rearranged AML. ARIH2 encodes TRIAD1, an E3 ubiquitin ligase required for termination of emergency granulopoiesis and leukemia suppressor function in MLL1-rearranged AML. Receptor tyrosine kinases (RTKs), including the FGF receptor, are TRIAD1 substrates that are possibly relevant to these activities. Using transcriptome analysis, we found increased activity of innate immune response pathways and RTK signaling in bone marrow progenitors from mice with MLL1-rearranged AML. We hypothesized that sustained RTK signaling, because of decreased TRIAD1 activity, impairs termination of emergency granulopoiesis during the innate immune response and contributes to leukemogenesis in this AML subtype. Consistent with this, we found aberrantly sustained emergency granulopoiesis in a murine model of MLL1-rearranged AML, associated with accelerated leukemogenesis. Treating these mice with an inhibitor of TRIAD1-substrate RTKs terminated emergency granulopoiesis, delayed leukemogenesis during emergency granulopoiesis, and normalized innate immune responses when combined with chemotherapy. Emergency granulopoiesis also hastened postchemotherapy relapse in mice with MLL1-rearranged AML, but remission was sustained by ongoing RTK inhibition. Our findings suggest that the physiological stress of infectious challenges may drive AML progression in molecularly defined subsets and identify RTK inhibition as a potential therapeutic approach to counteract this process.

Investigating the role of the innate immune response in relapse or blast crisis in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is characterized by expression of the tyrosine kinase oncogene, Bcr-abl. Tyrosine kinase inhibitors (TKI) induce prolonged remission in CML, and therapy discontinuation is an accepted approach to patients with reduction in Bcr-abl transcripts of four logs or greater. Half such individuals sustain a therapy free remission, but molecular mechanisms predicting relapse are undefined. We found relative calpain inhibition in CML cells with stabilization of calpain substrates, including ßcatenin and Xiap1. Since the Survivin gene is activated by ßcatenin, this identified two apoptosis-resistance mechanisms. We found that Survivin impaired apoptosis in leukemia stem cells (LSCs) and Xiap1 in CML granulocytes. Consistent with this, we determined treatment with an inhibitor of Survivin, but not Xiap1, prevented relapse during TKI treatment and after therapy discontinuation in a murine CML model. By transcriptome profiling, we identified activation of innate immune response pathways in murine CML bone marrow progenitors. This was increased by TKI treatment alone, but normalized with addition of a Survivin inhibitor. We found that activation of the innate immune response induced rapid blast crisis in untreated CML mice, and chronic phase relapse during a TKI discontinuation attempt. These results suggest that extrinsic stress exerts adverse effects on CML-LSCs.

Levels of angiogenic markers in second-trimester maternal serum from in vitro fertilization pregnancies with oocyte donation.

OBJECTIVE: To determine whether differences exist in angiogenic placental growth factor (PlGF) and antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR-1; both being early markers of placental ischemic disease) in oocyte-donation (OD) pregnancies, compared with autologous in vitro fertilization (aIVF) and spontaneous pregnancies. DESIGN: Case-control study of residual second-trimester serum samples from women undergoing prenatal screening. SETTING: Academic medical center. PATIENT(S): Fifty-seven OD pregnancies were identified. Each OD pregnancy was matched to two spontaneous pregnancies (n = 114) and one aIVF pregnancy (n = 57). INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): Second-trimester serum PlGF and sVEGFR-1 levels. RESULT(S): sVEGFR-1, PlGF, and unconjugated E2 levels were similar among the three study groups. The ratio of sVEGFR-1 to PlGF was significantly higher in the OD group. Consistently with previous studies, alpha-fetoprotein (AFP) in the OD group was significantly elevated compared with spontaneous pregnancy. Both aIVF and OD groups had greater levels of inhibin A than the spontaneous pregnancy group, and the OD group had significantly higher levels of inhibin A than the aIVF group. hCG levels were significantly elevated in aIVF compared with spontaneous pregnancy; however, levels were not different between aIVF and OD. CONCLUSION(S): Second-trimester serum sVEGFR-1 and PlGF levels were not significantly altered in OD pregnancies. Our data support previous findings that OD pregnancies have uniquely increased second-trimester AFP, hCG, and inhibin A levels compared with aIVF. However, the biologic basis of these marker elevations in OD may not be related to placental angiogenesis.

Multiple biomarker algorithms to predict epithelial ovarian cancer in women with a pelvic mass: Can additional makers improve performance?

INTRODUCTION: Management of a woman with a pelvic mass is complicated by difficulty in discriminating malignant from benign disease. Many serum biomarkers have been examined to determine their sensitivity for detecting malignancy. This study was designed to evaluate if the addition of biomarkers to HE4 and CA125, as used in the Risk of Malignancy Algorithm (ROMA), can improve the detection of EOC. METHODS: This was an IRB approved, prospective clinical trial examining serum obtained from women diagnosed with a pelvic mass who subsequently underwent surgery. Serum biomarker levels for CA125, HE4, YKL-40, transthyretin, ApoA1, Beta-2-microglobulin, transferrin, and LPA were measured. Logistic regression analysis was performed for various marker combinations, ROC curves were generated, and the area under the curves (AUCs) were determined. RESULTS: A total of 184 patients met inclusion criteria with a median age of 56 years (Range 20-91). Final pathology revealed there were 103 (56.0%) benign tumors, 4 (2.2%) LMP tumors, 61 EOC (33.1%), 2 (1.1%) non-EOC ovarian cancers, 6 (3.3%) gynecologic cancers with metastasis to the ovary and 8 (4.3%) non-gynecologic cancers with metastasis to the ovary. The combination of HE4 and CA125 (i.e. ROMA) achieved an AUC of 91.2% (95% CI: 86.0-96.4) for the detection of EOC vs benign disease. The combination of CA125, HE4, YKL-40, transthyretin, ApoA1, Beta 2 microglobulin, transferrin, LPA and menopausal status achieved the highest AUC of 94.6% (95% CI: 90.1-99.2) but this combination was not significantly better than the HE4 and CA125 combination alone (p = 0.078). CONCLUSIONS: The addition of select further serum biomarkers to HE4 and CA125 does not add to the performance of the dual marker combination for the detection of ovarian cancer.

Maternal BMI, Peripheral Deiodinase Activity, and Plasma Glucose: Relationships Between White Women in the HAPO Study.

OBJECTIVES: Explore the maternal body mass index (BMI) relationship with peripheral deiodinase activity further. Examine associations between deiodinase activity, glucose, and C-peptide. Consider findings in the historical context of related existing literature. DESIGN: Identify fasting plasma samples and selected demographic, biophysical, and biochemical data from a subset of 600 randomly selected non-Hispanic white women recruited in the Hyperglycemia Adverse Pregnancy Outcomes (HAPO) study, all with glucose tolerance testing [545 samples sufficient to measure TSH, free T4 (fT4), and T3]. Exclude highest and lowest 1% TSH values (535 available for analysis). Assess deiodinase activity by using T3/fT4 ratios. Among women with and without gestational diabetes mellitus (GDM), compare thyroid measurements, C-peptide, and other selected data. Examine relationships independent of GDM status between BMI and thyroid hormones and between thyroid hormones and glucose and C-peptide. RESULTS: Levels of BMI, T3/fT4 ratio, and T3 were significantly higher among women with GDM (P = 0.01, 0.005, and 0.001, respectively). Irrespective of GDM status, maternal BMI was associated directly with both T3/fT4 ratio (r = 0.40, P < 0.001) and T3 (r = 0.34, P < 0.001) but inversely with fT4 (r = -0.21, P < 0.001). In turn, fasting thyroid hormone levels (most notably T3/fT4 ratio) were directly associated with maternal glucose [z score sum (fasting, 1, 2 hours); r = 0.24, P < 0.001] and with C-peptide [z score sum (fasting, 1 hour); r = 0.27, P < 0.001]. CONCLUSIONS: Higher BMI was associated with increased deiodinase activity, consistent with reports from elsewhere. Increased deiodinase activity, in turn, was associated with higher glucose. Deiodinase activity accounts for a small percentage of z score sum glucose.

Inhibitory effects of SEL201 in acute myeloid leukemia.

MAPK interacting kinase (MNK), a downstream effector of mitogen-activated protein kinase (MAPK) pathways, activates eukaryotic translation initiation factor 4E (eIF4E) and plays a key role in the mRNA translation of mitogenic and antiapoptotic genes in acute myeloid leukemia (AML) cells. We examined the antileukemic properties of a novel MNK inhibitor, SEL201. Our studies provide evidence that SEL201 suppresses eIF4E phosphorylation on Ser209 in AML cell lines and in primary patient-derived AML cells. Such effects lead to growth inhibitory effects and leukemic cell apoptosis, as well as suppression of leukemic progenitor colony formation. Combination of SEL201 with 5'-azacytidine or rapamycin results in synergistic inhibition of AML cell growth. Collectively, these results suggest that SEL201 has significant antileukemic activity and further underscore the relevance of the MNK pathway in leukemogenesis.

Identification and targeting of novel CDK9 complexes in acute myeloid leukemia.

Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.

Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity.

Megakaryocyte (MK) migration from the bone marrow periosteal niche toward the vascular niche is a prerequisite for proplatelet extension and release into the circulation. The mechanism for this highly coordinated process is poorly understood. Here we show that dynasore (DNSR), a small-molecule inhibitor of dynamins (DNMs), or short hairpin RNA knockdown of DNM2 and DNM3 impairs directional migration in a human MK cell line or MKs derived from cultured CD34+ cells. Because cell migration requires actin cytoskeletal rearrangements, we measured actin polymerization and the activity of cytoskeleton regulator RhoA and found them to be decreased after inhibition of DNM2 and DNM3. Because SDF-1α is important for hematopoiesis, we studied the expression of its receptor CXCR4 in DNSR-treated cells. CXCR4 expression on the cell surface was increased, at least partially because of slower endocytosis and internalization after SDF-1α treatment. Combined inhibition of DNM2 and DNM3 or forced expression of dominant-negative Dnm2-K44A or GTPase-defective DNM3 diminished ß1 integrin (ITGB1) activity. DNSR-treated MKs showed an abnormally clustered staining pattern of Rab11, a marker of recycling endosomes. This suggests decreased recruitment of the recycling pathway in DNSR-treated cells. Altogether, we show that the GTPase activity of DNMs, which governs endocytosis and regulates cell receptor trafficking, exerts control on MK migration toward SDF-1α gradients, such as those originating from the vascular niche. DNMs play a critical role in MKs by triggering membrane-cytoskeleton rearrangements downstream of CXCR4 and integrins.