Search

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.

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.

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.

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.

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 β-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.

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 βcatenin and Xiap1. Since the Survivin gene is activated by βcatenin, 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.

Fanconi Anemia (FA) is an inherited disorder of DNA repair with hematologic manifestations that range from anemia to bone marrow failure (BMF) to acute myeloid leukemia (AML). In a murine model of FA (Fancc(−/−) mice), we found BMF 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 primarily influenced 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.

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.

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.

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.

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.

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.

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.

Introduction Snoring, the symptom of partial airway obstruction during sleep, is a common complaint during pregnancy and is associated with adverse perinatal outcomes. Mechanisms underlying this association have not been studied. We investigated the relationship between snoring in pregnancy and maternal serum markers of feto-placental wellbeing. Methods We conducted a secondary analysis of a cross sectional study designed to investigate perinatal outcomes of sleep-disordered breathing. Women admitted for delivery were systematically selected and answered a questionnaire about snoring using the Multivariable Apnea Prediction Index. Participants who had screening markers measured were included and divided into snorers and non -snorers. Markers measured included first and second trimester Down syndrome screening markers, reported as multiples of the median (MoM). An additional analysis was performed with snorers categorized as acute or chronic snorers based on duration of snoring in relation to pregnancy. Results While significant differences were noted in co-morbid maternal medical conditions between snorers and non-snorers, there were no significant differences in the neonatal outcomes assessed between the two groups. No significant differences were noted in any of the first trimester (PAPP-A) or second trimester (AFP, uE3, hCG, inhibin-A) markers between snorers and non-snorers, p > 0.25. In addition, no significant differences in marker levels were noted between acute and chronic snorers. Conclusion Snoring is not associated with alterations in the markers of fetal or placental wellbeing tested here and suggests that there are alternative mechanisms underlying the association between snoring and adverse perinatal outcomes.

Fas associated phosphatase 1 (Fap1) is a ubiquitously expressed protein tyrosine phosphatase. Fap1 substrates include Fas and Gsk3β, suggesting a role in regulating cell survival. Consistent with this, increased Fap1 expression is associated with resistance to Fas or platinum induced apoptosis in some human colon cancer tumors or cell lines. In the current studies, we found that Fap1 expression was significantly greater in CD133+ colon cancer stem cells compared to CD133- tumor cells. PTPN13 promoter activity (encoding Fap1) was repressed by interferon regulatory factor 2 (irf2), and expression of Fap1 and Irf2 were inversely correlated in CD133+ or CD133- colon cancer cells. We determined that CD133+ cells were relatively resistant to Fas or oxaliplatin induced apoptosis, but this was reversed by Fap1-knockdown or a Fap1-blocking tripeptide (SLV). In a murine xenograft model of colon cancer, we found treatment with SLV peptide significantly decreased tumor growth and relative abundance of CD133+CD44+ cells; associated with increased phosphorylation of Fap1 substrates. SLV peptide also enhanced inhibitory effects of oxaliplatin on tumor growth and Fap1 substrate phosphorylation in this model. Our studies suggest that therapeutically targeting Fap1 may decrease persistence of colon cancer stem cells during treatment with platinum chemotherapy by activating Fap1 substrates. In a murine model of chronic myeloid leukemia, we previously determined that inhibition of Fap1 decreased persistence of leukemia stem cells during tyrosine kinase inhibitor treatment. Therefore, Fap1 may be a tissue agnostic target to increase apoptosis in malignant stem cells.

Interferon consensus sequence–binding protein (Icsbp) is required for terminating emergency granulopoiesis, an episodic event responsible for granulocyte production in response to infections and a key component of the innate immune response. Icsbp inhibits the expression of Stat3 and C/ebpβ, transcription factors essential for initiating and sustaining granulopoiesis, and activates transcription of Fanconi C (FANCC), a DNA repair protein. In prior studies, we noted accelerated bone marrow failure in Fancc−/− mice undergoing multiple episodes of emergency granulopoiesis, associated with apoptosis of bone marrow cells with unrepaired DNA damage. Additionally, we found increased expression of Fanconi C and F proteins during emergency granulopoiesis. These findings suggest that Icsbp protects the bone marrow from DNA damage by increasing activity of the Fanconi DNA repair pathway, but the mechanisms for FANCC activation during initiation of emergency granulopoiesis are unclear. In this study, we observed that Stat3 and C/ebpβ activate FANCC transcription and contribute to DNA repair. Our findings indicate that FancC expression is increased during Stat3- and C/ebpβ-induced initiation of emergency granulopoiesis by these transcription factors and is maintained through termination by Icsbp. Our work reveals that Stat3- and C/ebpβ-mediated FancC expression is a critical component for initiating and sustaining key innate immune responses.

Chromosomal translocations involving the MLL1 gene characterize a poor prognosis subset of acute myeloid leukemia (AML), referred to as 11q23-AML. Transcription of the HOXA9 and HOXA10 genes is enhanced in hematopoietic stem and progenitor cells in these leukemias. We previously found the ARIH2 gene was repressed by HoxA9 in myeloid progenitors, but activated by HoxA10 during granulopoiesis. ARIH2 encodes the Triad1 protein, an anti-proliferative E3 ubiquitin ligase. In the current study, we investigate the role of Triad1 in leukemogenesis induced by an MLL1 fusion protein (Mll-Ell). We found Mll-Ell increased expression of HoxA9, HoxA10, and Triad1 because HoxA9 represses only one of two ARIH2 cis elements that are activated by HoxA10. Although Triad1 antagonized the generally pro-proliferative effects of the Mll-Ell oncoprotein, we found blocking HoxA9 and HoxA10 phosphorylation shifted the balance to ARIH2 repression in Mll-Ell+ cells. We investigated the significance of these in vitro results in a murine bone marrow transplant model. We found Triad1 knockdown significantly shortened the latency to development of AML in mice transplanted with Mll-Ell-transduced bone marrow. And, Triad1 expression fell during the prolonged AML latency period in mice transplanted with bone marrow expressing Mll-Ell alone. Our studies identify Triad1 as a leukemia suppressor in 11q23-AML. This suggests defining relevant Triad1 substrates may indicate novel therapeutic targets in this disease.

The dynamins are a family of ubiquitously expressed proteins with GTPase activity and are known for their role in membrane remodeling and intracellular trafficking. However, their exact role within various hematopoietic lineages is incompletely understood. In humans, most of the clinical cases with cytopenia in Charcot-Marie-Tooth (CMT) disease due to dynamin-2 mutations are associated with neutropenia, and CMT patients may suffer from impaired wound healing. Of interest, pregnancy notably worsens CMT disease, possibly due to hormonal changes. Antiprogesterone treatment was successfully given in a CMT rat model, and similar treatment is being considered for human CMT. We previously reported that inhibiting dynamin (DNM) activity impairs migration capability in mature megakaryocytes. We obtained the conditional deletion of Dnm2 and targeted its deletion in hematopoietic tissues with the vav-cre murine strain. Homozygous deletion of Dnm2 in blood tissues appears embryonic lethal. None of the pups born showed a Vav-cre/Dnm-2 fl/fl genotype, whereas a third of the pups born had a Vav-cre/Dnm-2 fl/wt (Dnm2 het) genotype. Bone marrow cells from the heterozygous female mice (Dnm2 het) had 35% to 50% decreased Dnm2 expression in comparison with age-matched controls (CTRL). Evaluation of the complete blood counts demonstrated that Dnm2 het female mice developed leukopenia which was detected from 40 weeks of age (average granulocyte-monocyte counts: CTRL 532/mm3 vs. Dnm2 het 300/mm3; p=0.0164). Neutropenia was unequivocal at 65 weeks of age (average neutrophil counts, CTRL 700/mm3 vs. Dnm2 het 343/mm3, p=0.016). Dnm2 het showed a trend for higher platelet counts than controls, but this was non-statistically significant. Further analysis of hematopoietic lineage maturation by flow cytometry indicated that lineage-negative cells and granulocyte-monocyte progenitors were decreased in Dnm2 het mice (average bone marrow lineage-negative cells: CTRL 2.8x10E6 vs. Dnm2 het 1.97x10E6, p =0.0056; average granulocyte-monocyte progenitors: CTRL 1.35x10E6 vs. Dnm2 het 0.85x10e6, p=0.01), along with a relative increase of common lymphoid progenitors and of megakaryocyte/erythrocyte progenitors in the bone marrow. Immunohistochemical staining for mature neutrophils with Ly6G showed an overall decreased number of mature granulocytes in the bone marrow of Dnm2 het mice (average Ly6G-positive cells: CTRL 20% vs. Dnm2 het 29%, p=0.0026). A linear pattern of distribution of Ly6G positive bone marrow cells along blood vessels was observed in fewer mice in the Dnm2 het group than in the CTRL group (25% vs. 59%, p=0.02), indicating that the migration pattern within the bone marrow is altered in the Dnm2 het group (see Figure). In addition, Dnm2 het mice developed splenomegaly (average spleen weight: Dnm2 het 146 mg vs. CTRL 99 mg, p=0.006), which was secondary to a marked florid reactive germinal center hyperplasia. Some of the Dnm2 het mice, including 5 mice whose pregnancy occurred in middle-age (p=0.005 when comparing with CTRL or young Dnm2 het mice) and 2 non-pregnant older mice, showed physical signs of distress with markedly reduced activity, poor grooming, ruffled furs, and hunched posture. Both non-pregnant sick mice showed a marked decrease in Ly6G positive mature neutrophils at 0.3% of total marrow cells (Figure), and the bone marrow from one mouse was completely effaced by immature myeloid precursors, consistent with the development of acute myeloid leukemia. In addition, a third of Dnm2 hetmice showed no distress but displayed morphological bone marrow abnormalities including megakaryocytic dysmorphology. In summary, female mice with loss of Dnm2 in the hematopoietic compartment develop persistent neutropenia as they age, with decreased granulocyte progenitor production and with migration defects. These abnormalities are associated with a risk for developing megakaryocytic dysplasia, and acute myeloid leukemia. These findings might also suggest a mechanism for chronic idiopathic neutropenia, which has a predominance in middle-aged women. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and occurs with much greater frequency in adults. Compared to adults, children with CML tend to present with higher white blood cell counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML are due to unique molecular characteristics that differ from adult CML. To test this hypothesis, we compared the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy age-matched CD34+ cells. CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric healthy (n=10), and adult healthy (n=10) bone marrow samples. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes. Four comparisons were performed: (1) pediatric CML vs pediatric healthy, (2) adult CML vs adult healthy, (3) pediatric CML vs adult CML, and (4) pediatric healthy vs adult healthy. A False Discovery Rate of ≤ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes (DEGs) in each comparison. To identify potentially unique pathways based on DEG, pathway over-representation was calculated with either goana from the limma package or clueGO. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients. Median WBC counts were 273,000 and 143,000 in pediatric and adult patients respectively. A total of 1352 genes were differentially expressed in either adult or pediatric CML CD34+ cells compared to healthy CD34+ cells, 174 of which were expressed similarly in pediatric and adult CML CD34+ cells (54 up- and 120 down-regulated). There were 746 differentially expressed genes (325 up- and 421 down-regulated) in adult CML CD34+ cells compared to adult healthy CD34+ cells, and 432 differentially expressed genes (156 up- and 276 down-regulated) in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. In direct comparison of pediatric and adult CML CD34+ cells, 446 genes (270 up and 176 down) were dysregulated in pediatric CML CD34+ cells. Pathway analysis showed that Rho signaling pathway was downregulated in pediatric CML CD34+ cells and several genes in Rho pathway were uniquely dysregulated. ARHGAP27 and VAV2 were significantly upregulated in adult CML CD34+ cells by 3.7-fold (p=0.0453) and 11-fold (p=0.0072), respectively, compared to pediatric CML CD34+ cells. In addition, several genes involved in the NADPH oxidase pathway, one of the best-characterized Rho GTPase-regulated systems, were differently expressed in CML. NCF1, CYBB, and S100A8 were significantly upregulated in adult CML CD34+ cells by 4-fold (p=0.0045), 3.26-fold (p These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the molecular biology of CML across the ages will provide new insights into the pathogenesis of pediatric CML and potentially inform future treatment decisions. Disclosures Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy.

Objective: To assess the clinical utility of cell-free DNA (cfDNA)-based screening for aneuploidies offered through primary obstetrical care providers to a general pregnancy population. Methods: Patient educational materials were developed and validated and providers were trained. Serum was collected for reflexive testing of cfDNA failures. Providers and patients were surveyed concerning knowledge, decision making, and satisfaction. Pregnancy outcome was determined by active or passive ascertainment. Results: Between September 2014 and July 2015, 72 providers screened 2,691 women. The five largest participating practices increased uptake by 8 to 40%. Among 2,681 reports, 16 women (0.6%) were screen-positive for trisomy 21, 18, or 13; all saw genetic professionals. Twelve were confirmed (positive predictive value (PPV), 75%; 95% CI, 48–93%) and four were false-positives (0.15%). Of 150 failures (5.6%), 79% had a negative serum or subsequent cfDNA test; no aneuploidies were identified. Of 100 women surveyed, 99 understood that testing was optional, 96 had their questions answered, and 95 received sufficient information. Pretest information was provided by the physician/certified nurse midwife (55) or office nurse/educator (40); none was provided by genetic professionals. Conclusion: This first clinical utility study of cfDNA screening found higher uptake rates, patient understanding of basic concepts, and easy incorporation into routine obstetrical practices. There were no reported cases of aneuploidy among cfDNA test failures. Genet Med advance online publication 12 January 2017

Objective: Serum markers measured early in pregnancy have been associated with the later diagnosis of gestational diabetes mellitus (GDM). This study aims to explore the performance of a panel of first-trimester biochemical markers for the prediction of GDM. Methods: A case-control study was performed that included 12 women who developed GDM and 60 controls matched for maternal and gestational age at blood collection. Levels of pregnancy-associated plasma protein A (PAPP-A), soluble endoglin, pregnancy protein 13, and adiponectin (Adipo) were measured on residual sera used in first-trimester screening for Down syndrome. Data were analyzed by nonparametric methods. A receiver operating characteristic curve was used to calculate the detection rate (DR) obtained with a panel of significant predictors for GDM. Results: Multiples of the median values for Adipo and PAPP-A were significantly reduced in GDM cases versus matched controls. Combination of Adipo and PAPP-A yielded a DR of 63.6% at a false-positive rate of 10%. Addition of body mass index (BMI) to this panel increased DR to 72.7%. Conclusion: This study suggests that first-trimester screening with Adipo, PAPP-A, and BMI may effectively identify women at high risk for the development of GDM.

// Weiqi Huang 1, 2 , Ling Bei 1, 2 , Elizabeth E. Hjort 1, 2 and Elizabeth A. Eklund 1, 2 1 The Feinberg School at Northwestern University, Chicago, IL, USA 2 Jesse Brown VA Medical Center, Chicago, IL, USA Correspondence to: Elizabeth A. Eklund, email: e-eklund@northwestern.edu Keywords: leukemia, Calpain, apoptosis, myeloid, survivin Received: November 10, 2016 Accepted: March 27, 2017 Published: April 06, 2017 ABSTRACT Chronic Myeloid Leukemia (CML) is characterized by translocations between chromosomes 9 and 22, resulting in expression of Bcr-abl oncogenes. Although the clinical course of CML was revolutionized by development of Bcr-abl-directed tyrosine kinase inhibitors (TKIs), CML is not cured by these agents. Specifically, the majority of subjects relapsed in clinical trials attempting TKI discontinuation, suggesting persistence of leukemia stem cells (LSCs) even in molecular remission. Identifying mechanisms of CML-LSC persistence may suggest rationale therapeutic targets to augment TKI efficacy and lead to cure. Apoptosis resistance is one proposed mechanism. In prior studies, we identified increased expression of Growth Arrest Specific 2 (Gas2; a Calpain inhibitor) in Bcr-abl + bone marrow progenitor cells. A number of previously described Calpain substrates might influence apoptosis in CML, including βcatenin and the X-linked Inhibitor of Apoptosis Protein 1 (Xiap1). We previously found Gas2/Calpain dependent stabilization of βcatenin in CML, and increased expression of βcatenin target genes, including Survivin (also an IAP). In the current work, we investigate contributions of Survivin and Xiap1 to Fas-resistance in Bcr-abl + bone marrow cells. Inhibitors of these proteins are currently in clinical trials for other malignancies, but a role for either IAP in CML-LSC persistence is unknown.

Pregnancy is a risk factor for sleep disordered breathing, including obstructive sleep apnea (OSA). Progesterone, one of the key hormones in pregnancy, a known respiratory drive stimulant, increases ventilation and may protect against OSA. We aimed to examine the relationship between circulating progesterone and OSA, after accounting for body weight and gestational age.A case control study was conducted of pregnant women with OSA and those at low risk for the disorder. Cases were identified by ICD-9 code and review of medical record. Controls were identified if they scored zero (never) for snoring, apnea, and gasping on the multivariable apnea prediction index questionnaire immediately following delivery. Subjects with available stored first and/or second trimester residual serum samples were then included in this study and serum analyzed for progesterone. Raw progesterone levels were adjusted for the effects of gestational age and maternal weight.Twenty-seven cases and 64 controls with available serum were identified. Women with OSA had greater maternal weight and higher rates of related comorbidities, compared to controls. Progesterone levels correlated positively with gestational age and negatively with greater weight. Progesterone levels, adjusted for gestational age and maternal weight and expressed as multiples of median (MoM), were significantly lower in OSA cases compared to controls in both the first trimester (MoM = 0.71, confidence interval [95% CI] 0.60-0.83) relative to the MoM in controls of 1.00. In the second trimester levels were also lower in OSA cases (MoM = 0.84, 95% CI 0.73-0.96) compared to the MoM of 1.00 in controls.Progesterone levels, after accounting for weight and gestational age, were lower in women with OSA than controls. Progesterone may play a protective role against OSA.

PURPOSE: Pregnant women are particularly susceptible to sleep disordered breathing. Obstructive sleep apnea (OSA) in pregnancy is associated with poor pregnancy and fetal outcomes. Oxidative stress caused by intermittent hypoxemia and re-oxygenation may impact pregnancy health. We hypothesize that pregnant women with OSA have a pronounced oxidative stress profile. METHODS: A case-control study was performed to study oxidative stress markers in the serum of pregnant women with or without OSA. Patients with OSA were identified between 2003–2009. Contemporaneous controls were pregnant subjects without apnea, gasping or snoring around the time of delivery. Serum markers of oxidative and carbonyl stress were measured by spectrophotometric/fluorometric methods. Multiple linear regression analysis was used with a model including age, body mass index at delivery, history of diabetes and gestational age. RESULTS: Serum samples from 23 OSA cases and 41 controls were identified. Advanced oxidation protein products (AOPP), a marker for oxidative stress, and advanced glycation end products (AGEs), a marker for carbonyl stress, were significantly lower in women with OSA than in controls (p-value< 0.0001). Total antioxidant capacity (TAC) was higher in women with OSA in comparison to controls (p-value

The inhibin alpha subunit protein is used in the histopathologic diagnosis of granulosa cell tumors (GCTs), and as a serum marker for disease progression. Yet, the availability of antibodies for inhibin has been limited. Serum antimüllerian hormone (AMH) levels have also been described as a GCT marker. The goal of this study was to compare inhibin and AMH immunoreactivity in tissues and serum from GCT (n=6) using existing and new antibodies. Expression was also explored in cases of mucinous tumors (n=15), where inhibin is also a serum marker in some cases. Immunocytochemistry was performed using a commercial and newly developed inhibin alpha subunit and AMH antibodies. Serum levels were examined with total inhibin and AMH immunoassays. Inhibin alpha subunit and AMH were equivalent markers of GCT in both tissue and serum. In mucinous samples, inhibin alpha subunit was detected in tumor and stromal cells, and levels in serum were also frequently elevated. In contrast, AMH protein was detected in mucinous tissues, but there was no evidence of secretion in serum. The new inhibin alpha subunit and AMH antibodies provide needed resources for examination of granulosa cell and mucinous tumors.

Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid leukemias and 2-9% of all pediatric leukemias. There are several unique characteristics of CML diagnosed in children, adolescents, and young adults, compared to adults. They present with higher white blood counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that differ from adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric normal (n=10) and adult normal (n=10) bone marrow samples. Total RNA was isolated from cells, and cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values (TMM) from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes (LogFC). We performed three comparisons: (1) Pediatric CML vs Normal, (2) Adult CML vs Normal, and (3) Pediatric CML vs Adult CML. A False Discovery Rate (FDR) of £ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes in each comparison. Over-representation analysis was used to identify potentially unique pathways based on differentially expressed genes. Clinical and demographic features at diagnosis were extracted for pediatric and adult CML patients and compared using Fisher's exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). Results Pediatric patients were diagnosed with CML at a median of 11 years (interquartile range (IQR): 10-14) compared to 54 years (IQR: 33-62) for adult patients. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients, whereas the white blood cell count and phase at diagnosis did not differ. We found 606 genes (210 up- and 396 down-regulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric normal controls. Interestingly, transcriptional regulators involved in blood cell differentiation including GATA1, TAL1, and KLF1 were differentially enriched in pediatric CML. In comparing adult CML patients to normal adult CD34+ cells, we found 920 genes (379 up- and 541 down-regulated) differentially expressed. Among all dysregulated genes we identified (1352 genes), 174 genes (54 up- and 120-down-regulated) overlapped when comparing pediatric and adult CML patients. Significantly enriched pathways in both adult and pediatric CML cells included PI3K/AKT signaling, MAPK signaling, and Notch/Wnt signaling, which have been previously reported. We found 437 unique genes that were dysregulated only in pediatric CML (270 up- and 167 down-regulated). Notch/Wnt signaling and Rho signaling pathways were significantly enriched. DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, has been known to be downregulated in solid tumors and hematologic malignancies. Interestingly, our data showed that DLC1 is significantly upregulated by 3-fold (p=0.0238) in pediatric CML, but not adult CML CD34+ cells. In addition, we observed that ABR, an inducer of C/EBPa that encodes an activator of RhoGEF and GTPase, was significantly downregulated by 2-fold (p=0.0119) in pediatric but not in adult CML CD34+ cells. Conclusion These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the particular biology of pediatric CML might impact the treatment of those patients in the future. Disclosures Gotlib: Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding.

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.

// Elizabeth E. Hjort 1, 2 , Weiqi Huang 1, 2 , Liping Hu 1, 2 , Elizabeth A. Eklund 1, 2 1 The Feinberg School at Northwestern University, Chicago, IL, USA 2 Jesse Brown VA Medical Center, Chicago, IL, USA Correspondence to: Elizabeth A. Eklund, email: e-eklund@northwestern.edu Keywords: leukemia, calpain, transcription factor, myeloid, phosphatase Received: August 01, 2016 Accepted: October 12, 2016 Published: October 19, 2016 ABSTRACT Icsbp/Irf8 is an interferon regulatory transcription factor that functions as a suppressor of myeloid leukemias. Consistent with this activity, Icsbp represses a set of genes encoding proteins that promote cell proliferation/survival. One such gene encodes Gas2, a calpain inhibitor. We previously found that increased Gas2-expression in Bcr-abl + cells stabilized βcatenin; a Calpain substrate. This was of interest, because βcatenin contributes to disease progression in chronic myeloid leukemia (CML). Calpain has additional substrates implicated in leukemogenesis, including Stat5. In the current study, we hypothesized that Stat5 activity in CML is regulated by Gas2/Calpain. We found that Bcr-abl-induced, Shp2-dependent dephosphorylation of Icsbp impaired repression of GAS2 by this transcription factor. The consequent decrease in Calpain activity stabilized Stat5 protein; increasing the absolute abundance of both phospho and total Stat5. This enhanced repression of the IRF8 promoter by Stat5 in a manner dependent on Icsbp, Gas2 and Calpain, but not Stat5 tyrosine phosphorylation. During normal myelopoiesis, increased expression and phosphorylation of Icsbp inhibits Calpain. In contrast, constitutive activation of Shp2 in Bcr-abl + cells impairs regulation of Gas2/Calpain by Icsbp, aberrantly stabilizing Stat5 and enhancing IRF8 repression. This novel feedback mechanism enhances leukemogenesis by increasing Stat5 and decreasing Icsbp. Bcr-abl targeted tyrosine kinase inhibitors (TKIs) provide long term disease control, but CML is not cured by these agents. Our studies suggest targeting Calpain might be a rational therapeutic approach to decrease persistent leukemia stem cells (LSCs) during TKI-treatment.

The transcription factor HOXA10 is an important regulator of myelopoiesis. Engineered over-expression of Hoxa10 in mice results in a myeloproliferative disorder that progresses to acute myeloid leukaemia (AML) over time, and in humans over-expression is associated with poor outcomes in AML. Here, we report that loss of Hoxa10 expression in mice results in reduced platelet count and platelet production, but does not affect clotting efficiency. About 40% fewer platelets were found in Hoxa10 null animals in comparison to wild type littermates. We found a nearly 50% reduction in the percentage of reticulated platelets in Hoxa10 null mice, suggesting deficient platelet production. Furthermore, Hoxa10 null animals recovered less efficiently from induced thrombocytopenia, supporting our hypothesis of defective platelet production. This also correlated with reduced colony formation potential of stem and progenitor cells seeded in megakaryocyte-enhancing conditions in vitro. Together, our results indicate that HOXA10 is important for megakaryopoiesis and platelet biogenesis.

// Bryan Mitton 1 , Katie Hsu 1 , Ritika Dutta 1 , Bruce C. Tiu 1 , Nick Cox 2 , Kevin G. McLure 1 , Hee-Don Chae 1 , Mark Smith 2 , Elizabeth A. Eklund 3 , David E. Solow-Cordero 4, * , Kathleen M. Sakamoto 1, * 1 Department of Pediatrics, Stanford University, Stanford, CA, USA 2 Medicinal Chemistry Knowledge Center, Stanford ChEM-H, Stanford, CA, USA 3 Division of Hematology/Oncology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, USA 4 High-Throughput Bioscience Center, Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA * These authors have contributed equally and share senior authorship Correspondence to: Kathleen M. Sakamoto, e-mail: kmsakamo@stanford.edu Keywords: small molecule screen, novel therapeutics, CREB Received: September 25, 2015 Accepted: January 13, 2016 Published: January 30, 2016 ABSTRACT The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ~114,000 candidate compounds from Stanford University’s small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML.

e18074 Background: Both CA125 and HE4 are biomarkers used to monitor women diagnosed with epithelial ovarian cancer (EOC). When these biomarkers increase and suggest progression of disease, imaging is frequently obtained for further assessment and confirmation. This study aims to assess how far in advance these biomarkers begin to trend upwards in comparison to the radiographic evidence of disease progression. Methods: This was an IRB approved retrospective trial. Residual longitudinal serum samples drawn for serum CA125 levels during treatment and monitoring from women with EOC were obtained. The samples were drawn every 3-5-weeks for patients on chemotherapy and every 3 months while monitoring. Serum CA125 and HE4 levels in the residual sera were analyzed at each time point. Imaging data was collected for each patient. Patients were included if either HE4 or CA125 was a marker for their disease. For each patient, the date of time point when the biomarker started to trend upward was identified and the date of radiographic evidence of progression was determined. The lead time (in days) for elevation of both CA125 and HE4 prior to evidence of radiographic progression was determined for each patient. Results: A total of 48 patients undergoing 58 treatment and monitoring events were identified. Each had evidence of disease progression by CA125 levels, HE4 levels, and computed tomography (CT). CA125 was the first biomarker to trend upward for 9 events (15.5%), HE4 was the first biomarker for 10 events (17.2%), and both increased simultaneously for 39 events (67.2%). Mean lead time for CA125 elevation prior to progression on imaging was 60 days (95% CI: 43 - 78, median = 44, range = 0 - 386). For HE4, mean lead time was 66 days (95% CI: 48 – 83, median = 46, range = 0 - 386). There was no significant difference in the average lead times between the two biomarkers (p < 0.01). Conclusions: When used for EOC monitoring both CA125 and HE4 have similar average lead times (~2 months) prior to the detection of progressive or recurrent disease by CT imaging.

Emergency (stress) granulopoiesis is an episodic process for the production of granulocytes in response to infectious challenge. We previously determined that Fanconi C, a component of the Fanconi DNA-repair pathway, is necessary for successful emergency granulopoiesis. Fanconi anemia results from mutation of any gene in this pathway and is characterized by bone marrow failure (BMF) in childhood and clonal progression in adolescence. Although murine Fanconi anemia models exhibit relatively normal steady-state hematopoiesis, FANCC−/− mice are unable to mount an emergency granulopoiesis response. Instead, these mice develop BMF and die during repeated unsuccessful emergency granulopoiesis attempts. In FANCC−/− mice, BMF is associated with extensive apoptosis of hematopoietic stem and progenitor cells through an undefined mechanism. In this study, we find that TP53 haploinsufficiency completely rescues emergency granulopoiesis in FANCC−/− mice and protects them from BMF during repeated emergency granulopoiesis episodes. Instead, such recurrent challenges accelerated clonal progression in FANCC−/−TP53+/− mice. In FANCC−/− mice, BMF during multiple emergency granulopoiesis attempts was associated with increased ataxia telangiectasia and Rad3-related protein (Atr) and p53 activation with each attempt. In contrast, we found progressive attenuation of expression and activity of Atr, and consequent p53 activation and apoptosis, in the bone marrow of FANCC−/−TP53+/− mice during this process. Therefore, activation of Atr—with consequent Fanconi-mediated DNA repair or p53-dependent apoptosis—is an essential component of emergency granulopoiesis and it protects the bone marrow from genotoxic stress during this process.

Objectives Examine primary Down syndrome screening using combinations of first trimester serum markers, with and without sequencing of cell free DNA as a secondary reflexive test. Methods Samples from 40 Down syndrome cases were matched with five control samples and tested for PAPP-A, free β, AFP, inhibin-A and PlGF. Results were converted to weight-adjusted multiples of the median (MoM) and population parameters computed. Monte Carlo simulation modeled Down syndrome detection and false positive rates for various marker combinations. After reflexive DNA testing, the revised detection and false positive rates were also computed. Results At a primary false positive rate of 20%, the baseline combination (maternal age, PAPP-A and free β) detected 86.9%. Adding AFP or PlGF increased detection to 89.8% and 89.5%, respectively. Adding AFP and PlGF, AFP and inhibin-A, or all three markers, detected 93.7%, 94.1% and 95.5%, respectively. Modeling reflexive cf DNA testing results in little loss in detection (1%), but false positive rates fall to 0.2%. Conclusion First trimester reflexive testing does not require nuchal translucency measurements, and has high detection and very low rates of invasive procedures. However, timing of DNA sample collection and the costs of sample collection and DNA testing need to be considered before implementation. © 2015 John Wiley & Sons, Ltd.

Expression of the E3 ubiquitin ligase Triad1 is greater in mature granulocytes than in myeloid progenitor cells. HoxA10 actives transcription of the gene encoding Triad1 (ARIH2) during myeloid differentiation, but the contribution of increased Triad1 expression to granulocyte production or function is unknown. Mice with bone marrow–specific disruption of the ARIH2 gene exhibit constitutive inflammation with tissue infiltration by granulocytes and B cells. In contrast, disruption of the HOXA10 gene in mice neither constitutively activates the innate immune response nor significantly alters steady-state granulopoiesis. This study explores the impact of HoxA10-induced Triad1 expression on emergency (stress) granulopoiesis. We found that mice with HOXA10 gene disruption exhibited an overwhelming and fatal emergency granulopoiesis response that was characterized by tissue infiltration with granulocytes, but reversed by re-expression of Triad1 in the bone marrow. We determined that HoxA9 repressed ARIH2 transcription in myeloid progenitor cells, antagonizing the effect of HoxA10 on Triad1 expression. Also, we found that differentiation-stage–specific ARIH2 transcription was regulated by the tyrosine phosphorylation states of HoxA9 and HoxA10. Our studies demonstrate a previously undescribed role for HoxA10 in terminating emergency granulopoiesis, suggesting an important contribution by Hox proteins to the innate immune response.

Preeclampsia is a serious complication of pregnancy, threatening fetal and maternal health. The aim of our study is to examine the association between preeclampsia and the connecting peptide of the pregnancy hormone relaxin (pro-RLX2) as a potential new biochemical marker.This is a nested case/control study derived from the cohort of pregnancies delivering at WomenInfants Hospital. Cases were identified at a clinic or by hospital codes, and individually confirmed by record review. Stored samples were available from 'integrated' Down syndrome screening. Results were expressed as multiples of the median (MoM).Preeclampsia was classified as early/severe, late/severe, or mild based on professional guidelines.Fifty-one cases were each matched with five control pregnancies. Population distribution parameters were derived for cases and controls. As shown previously, discrimination between cases and controls (applying MoM analysis) was possible for PlGF (0.576, p .05), inhibin A (1.45, p .05) and endoglin (1.278, p .05). No association with preeclampsia was found for pro-RLX2. However, pro-RLX2 correlates with Inhibin A and Endoglin.Endoglin, Inhibin A and PlGF are highly predictive of preeclampsia. Quantification of pro-RLX2 is not able to predict preeclampsia. Nevertheless, the potential involvement of relaxin 2/pro-RLX2 in the pathophysiology of preeclampsia requires further study.

Objectives To determine whether maternal plasma collected in cell-free DNA stabilizing tubes is suitable for measuring prenatal screening ‘serum’ markers. Methods Matched plasma and serum samples were collected from 41 second trimester and 42 first trimester non-Down’s syndrome pregnancies. Second trimester samples were tested for alpha-fetoprotein, unconjugated estriol, human chorionic gonadotropin, and inhibin-A (Beckman Coulter DxI immunoassay). First trimester samples were tested for human chorionic gonadotropin and pregnancy-associated plasma protein A. Method comparisons performed for each marker compared plasma and serum results. Down’s syndrome likelihood ratios in serum and plasma were compared. Results Plasma and serum results for all markers were highly correlated ( r > 0.983) but for all, plasma results differed, usually by proportional amounts. After conversion to multiples of the median using sample type-specific medians, the logarithmic standard deviations in serum and plasma did not differ (all p > 0.37). Likelihood ratios for the first and second trimester marker combinations were highly correlated and closely agreed (log likelihood ratios range 1.005 to 1.032; 1.000 indicates complete agreement). Conclusions These results using specialized plasma collection tubes are similar to those of our earlier study showing that plasma collected in EDTA tubes is suitable for ‘serum’ Down’s syndrome screening. Laboratories must account for proportional changes by computing new plasma medians or modifying existing serum medians. Using a portion of the plasma from cell-free DNA collection tubes for ‘serum screening’ may have an advantage in programmes that are reflexively testing cell-free DNA, as only one sample type need be collected.

BackgroundHigh maternal weight is known to associate with both low free thyroxine and gestational diabetes mellitus. We explore a deiodinase-related mechanism that may help explain these associations.MethodsAmong 108 women receiving routine oral glucose tolerance testing for gestational diabetes mellitus, we collected biophysical data and measured free thyroxine and total triiodothyronine, using residual plasma samples.ResultsFasting triiodothyronine/free thyroxine ratio and triiodothyronine were higher among women with gestational diabetes mellitus ( p = 0.02; p = 0.04). The triiodothyronine/free thyroxine ratio and triiodothyronine measurements at 2 h were associated with weight ( r = 0.20, p = 0.04; r = 0.22, p = 0.02); free thyroxine showed a non-significant inverse weight relationship ( r = −0.06, p = 0.55). Glucose at all four intervals was associated with triiodothyronine/free thyroxine ratios, and triiodothyronine at 2 h. In stepwise regression, triiodothyronine/free thyroxine ratio predicted glucose more strongly than did weight.ConclusionThese relationships may be explained by higher maternal weight inducing peripheral deiodinase activity, resulting in higher plasma glucose (via triiodothyronine stimulation) and thereby increasing gestational diabetes mellitus risk.

Background Measurement of antimüllerian hormone (AMH) is used to assess ovarian reserve. Circulating levels of AMH correlate with antral follicle count, with relatively high levels indicating an ample reserve of primary and preantral follicles in the ovary. AMH levels are stable with dilution and freezer storage, and are not altered by hemolysis or menstrual cycle day in young women of reproductive age. We sought to examine whether glucose challenge or food intake modifies AMH levels compared with fasting. Methods Residual plasma samples were available from 54 pregnant women under fasting conditions and then 1, 2, and 3 h after ingestion of a 100-g glucose challenge. These samples were collected as part of routine clinical care to identify gestational diabetes (GDM) at 24–28 weeks of gestation. Twelve of these women met criteria for GDM based on an increased glucose level at a minimum of 2 time points. A second set consisted of serum samples collected from 8 nonpregnant women at fasting and 1 h after a meal. Levels of AMH were measured using an ultrasensitive assay (Ansh Labs, Webster, TX). A 2-way ANOVA (sample timing and GDM status) or matched t-test was performed. AMH measurements were subject to a logarithmic transformation before analysis. Results Median AMH levels in pregnant women at 1, 2, or 3 h after glucose challenge did not differ compared with AMH levels at fasting or by diagnosis of GDM. Similarly, there was no difference in median AMH levels in nonpregnant women of reproductive age at fasting and after a meal. Conclusion AMH levels are not altered by glucose or food intake.

The precise signaling mechanisms by which type II IFN receptors control expression of unique genes to induce biological responses remain to be established. We provide evidence that Sin1, a known element of the mammalian target of rapamycin complex 2 (mTORC2), is required for IFNγ-induced phosphorylation and activation of AKT and that such activation mediates downstream regulation of mTORC1 and its effectors. These events play important roles in the assembly of the eukaryotic translation initiation factor 4F (eIF4F) and mRNA translation of IFN-stimulated genes. Interestingly, IFNγ-induced tyrosine phosphorylation of STAT1 is reduced in cells with targeted disruption of Sin1, leading to decreased transcription of several IFNγ-inducible genes in an mTORC2-independent manner. Additionally, our studies establish that Sin1 is essential for generation of type II IFN-dependent antiviral effects and antiproliferative responses in normal and malignant hematopoiesis. Together, our findings establish an important role for Sin1 in both transcription and translation of IFN-stimulated genes and type II IFN-mediated biological responses, involving both mTORC2-dependent and -independent functions.

Preeclampsia is a serious complication of pregnancy, threatening fetal and maternal health. The aim of our study is to examine the association between preeclampsia and biochemical markers, in matched first and second trimester maternal serum samples.This is a nested case/control study derived from the cohort of pregnancies delivering at WomenInfants Hospital. Cases were identified at a clinic or by hospital codes, and individually confirmed by record review. Stored samples were available from 'integrated' Down syndrome screening. Results were expressed as multiples of the median (MoM).Preeclampsia was classified as early/severe, late/severe, or mild based on professional guidelines. An additional adverse outcome group had only gestational hypertension.Ninety-eight cases were each matched with five control pregnancies. Population distribution parameters and within and between trimester correlations were derived for cases and controls for six markers, as well as in case subgroups. The strongest associations were for early/severe preeclampsia with second trimester PAPP-A (rank sum test 2.30, p0.01); PlGF (2.60, p0.05) inhibin A (4.45, p0.05) and endoglin (4.25, p0.05). No strong associations were found for sVEGF-R and FLRG. Second trimester associations were stronger than those in the first (e.g., PAPP-A 2.45, p0.01). No between-trimester associations were found that would provide important improvements in prediction.This matched analysis of the serum markers in early pregnancy allows for direct comparison of first and second trimester associations with preeclampsia. PAPP-A and PlGF are equally and highly predictive of early/severe preeclampsia.

The subset of acute myeloid leukemias (AML) with chromosomal translocations involving the MLL gene have a poor prognosis (referred to as 11q23-AML). The MLL fusion proteins that are expressed in 11q23-AML facilitate transcription of a set of HOX genes, including HOXA9 and HOXA10. Because Hox proteins are transcription factors, this suggests the possibility that Hox target genes mediate the adverse effects of MLL fusion proteins in leukemia. Identifying such Hox target genes might provide insights to the pathogenesis and treatment of 11q23-AML. In the current study we found that Mll-Ell (an MLL fusion protein) induced transcriptional activation of the FGF2 gene in a HoxA9- and HoxA10-dependent manner. FGF2 encodes fibroblast growth factor 2 (also referred to as basic fibroblast growth factor). Fgf2 influences proliferation and survival of hematopoietic stem cells and myeloid progenitor cells, and increased Fgf2-expression has been described in AMLs. We determined that expression of Mll-Ell in myeloid progenitor cells resulted in autocrine production of Fgf2 and Fgf2-dependent cytokine hypersensitivity. Therefore, our results implicated increased Fgf2 expression in progenitor proliferation and expansion in 11q23-AML. Because small molecule inhibitors of Fgf-receptors are in human clinical trials, this suggested a potential therapeutic approach to this treatment refractory leukemia.

The mechanisms of generation of the antineoplastic effects of interferons (IFNs) in malignant hematopoietic cells remain to be precisely defined. We examined the activation of type I IFN-dependent signaling pathways in malignant cells transformed by Jak2V617F, a critical pathogenic mutation in myeloproliferative neoplasms (MPNs). Our studies demonstrate that during engagement of the type I IFN receptor (IFNAR), there is activation of Jak-Stat pathways and also engagement of Mnk kinases. Activation of Mnk kinases is regulated by the Mek/Erk pathway and is required for the generation of IFN-induced growth inhibitory responses, but Mnk kinase activation does not modulate IFN-regulated Jak-Stat signals. We demonstrate that for type I IFNs to exert suppressive effects in malignant hematopoietic progenitors from patients with polycythemia vera, induction of Mnk kinase activity is required, as evidenced by studies involving pharmacological inhibition of Mnk or siRNA-mediated Mnk knockdown. Altogether, these findings provide evidence for key and essential roles of the Mnk kinase pathway in the generation of the antineoplastic effects of type I IFNs in Jak2V617F-dependent MPNs.

Human epididymis protein 4 is a serum biomarker to aid in differentiating benign and malignant disease in women with a pelvic mass. Interpretation of human epididymis protein 4 results relies on robust normative data.The purpose of this study was to evaluate whether human epididymis protein 4 levels are variable in women during the normal menstrual cycle.Healthy women, 18-45 years old, with regular menstrual cycles were recruited from community gynecologic practices in Rhode Island. Women consented to enroll and to participate by the donation of blood and urine samples at 5 specific times over the course of each cycle. Levels of reproductive hormones and human epididymis protein 4 were determined. Data were analyzed with the use of linear regression after log transformation.Among 74 enrolled cycles, 53 women had confirmed ovulation during the menstrual cycle and completed all 5 sample collections. Levels of estradiol, progesterone, and luteinizing hormone displayed the expected menstrual cycle patterns. Levels of human epididymis protein 4 in serum were relatively stable across the menstrual cycle, except for a small ovulatory (median, 37.0 pM) increase. Levels of human epididymis protein 4 in urine, after correction for creatinine, displayed the same pattern of secretion observed in serum.Serum human epididymis protein 4 levels are relatively stable across the menstrual cycle of reproductive-aged women and can be determined on any day to evaluate risk of ovarian malignancy. A slight increase is expected at ovulation; but even with this higher human epididymis protein 4 level, results are well within the healthy reference range for women (120 pM). Levels of human epididymis protein 4 in urine warrant further investigation for use in clinical practice as a simple and convenient sample.