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Focusing on the issues of slow convergence speed and the ease of falling into a local optimum when optimizing the weights and thresholds of a back-propagation artificial neural network (BPANN) by the gradient method, a prediction method for pork supply based on an improved mayfly optimization algorithm (MOA) and BPANN is proposed. Firstly, in order to improve the performance of MOA, an improved mayfly optimization algorithm with an adaptive visibility coefficient (AVC-IMOA) is introduced. Secondly, AVC-IMOA is used to optimize the weights and thresholds of a BPANN (AVC-IMOA_BP). Thirdly, the trained BPANN and the statistical data are adopted to predict the pork supply in Heilongjiang Province from 2000 to 2020. Finally, to demonstrate the effectiveness of the proposed method for predicting pork supply, the pork supply in Heilongjiang Province was predicted by using AVC-IMOA_BP, a BPANN based on the gradient descent method and a BPANN based on a mixed-strategy whale optimization algorithm (MSWOA_BP), a BPANN based on an artificial bee colony algorithm (ABC_BP) and a BPANN based on a firefly algorithm and sparrow search algorithm (FASSA_BP) in the literature. The results show that the prediction accuracy of the proposed method based on AVC-IMOA and a BPANN is obviously better than those of MSWOA_BP, ABC_BP and FASSA_BP, thus verifying the superior performance of AVC-IMOA_BP.

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.

// 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.

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.

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.

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.

Ling Bei,1 Iris Lee,2 Michael S Lee,3 Greg P Van Stavern,1 Collin M McClelland3 1Department of Ophthalmology and Visual Neurosciences, Washington University School of Medicine, St Louis, MO, 2Casey Eye Institute, Oregon Health and Science University, Portland, OR, 3Department of Ophthalmology and Visual Neurosciences, University of Minnesota, Minneapolis, MN, USA Abstract: Giant-cell arteritis (GCA) is a visually devastating disease that often progresses to severe bilateral vision loss if untreated. Diagnosis of GCA is made challenging by the protean nature of the disease and the lack of a simple test that is both highly sensitive and specific. Choroidal filling delay on fluorescein angiography (FA) has been touted as a highly characteristic feature of GCA-related vision loss, although knowledge of both the sensitivity and specificity of this finding remains unproven. We report our experience of delayed choroidal filling on FA in a series of seven patients referred to an academic neuro-ophthalmology practice due to concern for GCA. Despite the FA findings, our examination, diagnostic testing, and long-term follow-up excluded the diagnosis of GCA in all cases, suggesting that choroidal perfusion abnormalities may occur in the absence of GCA. When evaluating a patient for acute vision loss, the astute clinician must remain cognizant of the limitations of FA in the diagnosis of GCA.Keywords: giant-cell arteritis, fluorescein angiography, delayed choroidal filling

// Chirag A. Shah 1 , Ling Bei 1, 2 , Hao Wang 1 , Jessica K. Altman 1 , Leonidas C. Platanias 1, 2 , Elizabeth A. Eklund 1, 2 1 The Feinberg School of Medicine and Robert H. Lurie Comprehensive Cancer Center of Northwestern University, Chicago, IL, USA 2 Jesse Brown Veteran’s Administration Medical Center, Chicago, IL, USA Correspondence to: Elizabeth A. Eklund, email: e-eklund@northwestern.edu Keywords: transcription factor, integrin, leukemia, homeobox, fibroblast growth factor Received: April 04, 2016 Accepted: June 03, 2016 Published: June 20, 2016 ABSTRACT A poor prognosis subtype of acute myeloid leukemia (AML) is characterized by increased expression of a set of homeodomain (HD) transcription factors, including HoxA9, HoxA10 and Cdx4. This encompasses AML with MLL1 gene translocations, because Mll1-fusion proteins aberrantly activate HOX transcription. We previously identified FGF2 (Fibroblast Growth Factor 2) as a target gene for HoxA9 and HoxA10 that was indirectly activated by Mll-Ell (an Mll1-fusion protein). Autocrine stimulation of Mll-Ell + myeloid progenitor cells by Fgf2 stabilized βcatenin and increased expression of βcatenin target genes, including CDX4 . Since HOXA9 and HOXA10 are Cdx4 target genes, Fgf2 indirectly augmented direct effects of Mll-Ell on these genes. ITGB3 , encoding β3 integrin, is another HoxA10 target gene. In the current studies, we found activation of ITGB3 transcription in Mll-Ell + myeloid progenitor cells via HoxA9 and HoxA10. Increased expression of αvβ3 integrin increased Syk-activation; contributing to cytokine hypersensitivity. However, inhibiting Fgf-R partly reversed αvβ3 activity in Mll-Ell + progenitor cells by decreasing ITGB3 promoter activity in a βcatenin- and Cdx4-dependent manner. Inhibitors of Fgf-R or Syk impaired proliferation of CD34 + bone marrow cells from AML subjects with increased Hox-expression; with a greater combined effect. These studies identified a rational therapeutic approach to this AML subtype.

The interferon consensus sequence binding protein (Icsbp) is a transcription factor that influences multiple aspects of myelopoiesis. Expression of Icsbp is decreased in the bone marrow of human subjects with chronic myeloid leukemia (CML), and studies in murine models suggest that Icsbp functions as an anti-oncogene for CML. We previously identified a set of Icsbp target genes that may contribute to this anti-oncogene effect. The set includes PTPN13, the gene encoding Fas-associated phosphatase 1 (Fap1, a Fas antagonist). We previously demonstrated that myeloid progenitor cells from Icsbp-knockout mice exhibit Fap1-dependent Fas resistance. In the present study, we determined that the Fas resistance of Bcr−abl+ cells is Icsbp- and Fap1-dependent. We also found that treatment of Bcr−abl+ bone marrow cells with a Fap1-blocking peptide prevents in vitro selection of a tyrosine kinase inhibitor (TKI)-resistant population. Therefore, these results have implications for therapeutic targeting of the Fas-resistant leukemia stem cell population and addressing TKI resistance in CML.

HoxA10 is a homeodomain transcription factor that is maximally expressed in myeloid progenitor cells. An increase in HoxA10 expression correlates with poor prognosis in human acute myeloid leukemia (AML). Consistent with this scenario, HoxA10 overexpression in murine bone marrow induces a myeloproliferative neoplasm that advances AML over time. Despite the importance of HoxA10 for leukemogenesis, few genuine HoxA10 target genes have been identified. The current study identified ARIH2, the gene encoding Triad1, as a HoxA10 target gene. We identified two distinct HoxA10-binding cis elements in the ARIH2 promoter and determined that HoxA10 activates these cis elements in myeloid cells. Triad1 has E3 ubiquitin ligase activity, and we found that HoxA10-overexpressing myeloid cells exhibited a Triad1-dependent increase in protein ubiquitination. Therefore, these studies have identified the regulation of protein ubiquitination as a novel function of Hox transcription factors. Forced overexpression of Triad1 has been show previously to inhibit colony formation by myeloid progenitor cells. In contrast, HoxA10-overexpressing myeloid progenitor cells exhibited increased proliferation in response to low doses of various cytokines. We found that Triad1 knockdown further increased cytokine-induced proliferation in HoxA10-overexpressing cells. Therefore, these studies have identified a HoxA10 target gene that antagonizes the overall influence of overexpressed HoxA10 on myeloproliferation. This result suggests that the consequences of HoxA10 overexpression reflect a balance between the target genes that facilitate and antagonize proliferation. These results have implications for understanding the mechanisms of leukemogenesis in AML with Hox overexpression.

Chronic myeloid leukemia (CML) is characterized by expression of Bcr-abl, a tyrosine kinase oncogene. Clinical outcomes in CML were revolutionized by development of Bcr-abl-targeted tyrosine kinase inhibitors (TKIs), but CML is not cured by these agents. CML leukemia stem cells (LSCs) are relatively TKI insensitive and persist even in remission. LSC persistence results in relapse upon TKI discontinuation, or drug resistance or blast crisis (BC) during prolonged treatment. We hypothesize that increased expression of Fas-associated phosphatase 1 (Fap1) in CML contributes to LSC persistence and BC. As Fap1 substrates include Fas and glycogen synthase kinase-3β (Gsk3β), increased Fap1 activity in CML is anticipated to induce Fas resistance and stabilization of β-catenin protein. Resistance to Fas-induced apoptosis may contribute to CML LSC persistence, and β-catenin activity increases during BC. In the current study, we directly tested the role of Fap1 in CML LSC persistence using in an in vivo murine model. In TKI-treated mice, we found that inhibiting Fap1, using a tripeptide or small molecule, prevented TKI resistance, BC and relapse after TKI discontinuation; all events observed with TKI alone. In addition, Fap1 inhibition increased Fas sensitivity and decreased β-catenin activity in CD34(+) bone marrow cells from human subjects with CML. Therapeutic Fap1 inhibition may permit TKI discontinuation and delay in progression in CML.

Emergency granulopoiesis occurs in response to infectious or inflammatory challenge and is a component of the innate immune response. Some molecular events involved in initiating emergency granulopoiesis are known, but termination of this process is less well defined. In this study, we found that the interferon consensus sequence binding protein (Icsbp/Irf8) was required to terminate emergency granulopoiesis. Icsbp is an interferon regulatory transcription factor with leukemia suppressor activity. Expression of Icsbp is decreased in chronic myeloid leukemia, and Icsbp(-/-) mice exhibit progressive granulocytosis with evolution to blast crisis, similar to the course of human chronic myeloid leukemia. In this study, we found aberrantly sustained granulocyte production in Icsbp(-/-) mice after stimulation of an emergency granulopoiesis response. Icsbp represses transcription of the genes encoding Fas-associated phosphatase 1 (Fap1) and growth arrest-specific 2 (Gas2) and activates genes encoding Fanconi C and F. After stimulation of emergency granulopoiesis, we found increased and sustained expression of Fap1 and Gas2 in bone marrow myeloid progenitor cells from Icsbp(-/-) mice in comparison with the wild type. This was associated with resistance to Fas-induced apoptosis and increased β-catenin activity in these cells. We also found that repeated episodes of emergency granulopoiesis accelerated progression to acute myeloid leukemia in Icsbp(-/-) mice. This was associated with impaired Fanconi C and F expression and increased sensitivity to DNA damage in bone marrow myeloid progenitors. Our results suggest that impaired Icsbp expression enhances leukemogenesis by deregulating processes that normally limit granulocyte expansion during the innate immune response.

Previous studies reported that characteristic lens opacities were present in Alzheimer Disease (AD) patients postmortem. We therefore determined whether cataract grade or lens opacity is related to the risk of Alzheimer dementia in participants who have biomarkers that predict a high risk of developing the disease. AD biomarker status was determined by positron emission tomography-Pittsburgh compound B (PET-PiB) imaging and cerebrospinal fluid (CSF) levels of Aβ42. Cognitively normal participants with a clinical dementia rating of zero (CDR=0; N=40) or with slight evidence of dementia (CDR=0.5; N=2) were recruited from longitudinal studies of memory and aging at the Washington University Knight Alzheimer's Disease Research Center. The age, sex, race, cataract type and cataract grade of all participants were recorded and an objective measure of lens light scattering was obtained for each eye using a Scheimpflug camera. Twenty-seven participants had no biomarkers of Alzheimer dementia and were CDR=0. Fifteen participants had biomarkers indicating increased risk of AD, two of which were CDR=0.5. Participants who were biomarker positive were older than those who were biomarker negative. Biomarker positive participants had more advanced cataracts and increased cortical light scattering, none of which reached statistical significance after adjustment for age. We conclude that cataract grade or lens opacity is unlikely to provide a non-invasive measure of the risk of developing Alzheimer dementia. Key Words: Lens opacity, cataract, Scheimpflug imaging, Alzheimer disease biomarkers, preclinical Alzheimer disease

Cdx and Hox proteins are homeodomain transcription factors that regulate hematopoiesis. Transcription of the HOX and CDX genes decreases during normal myelopoiesis, but is aberrantly sustained in leukemias with translocation or partial tandem duplication of the MLL1 gene. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decreased Cdx4 expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells, antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. As HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia-associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4 expression in myeloid progenitor cells. However, Cdx4 decreases in a HoxA9-dependent manner on exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia-associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison with myeloid progenitor cells that are expressing Mll-Ell alone, we find increased CDX4 transcription and Cdx4 expression in cells co-expressing Mll-Ell plus constitutively active Shp2. Increased Cdx4 expression is sustained on exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. This is clinically relevant, because MLL1 translocations and constitutive Shp2 activation co-exist in human myeloid leukemias.

It was investigated why the fMLP-stimulated respiratory burst in human neutrophils was enhanced by N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide (W-7), a considered calmodulin antagonist, at lower concentration but inhibited at higher concentration. Flow cytometric analysis on binding of the receptor to the fluorescence-labeled formyl peptide and the polymerization of actin in cells showed that the drug inhibited actin polymerization and promoted expression of the fMLP receptors on cell membrane at lower concentration, while promoted the actin polymerization and depressed the receptor expression at higher concentration. As intracellular Ca 2a ([Ca 2a ]i) is elevated, polymerization of actin decreases and the receptor expression increases. At normal physiological and two moderately high intracellular calcium levels, the dual effect of W-7 became less significant as [Ca 2a ]i was elevated indicating that the dual effect is calcium-dependent. Under two extreme conditions that the intracellular calcium was either depleted or highly elevated, the dual effect disappeared but only an inhibitory effect on actin polymerization was observed. Colchicine and taxol study showed that disruption or stabilization of microtubules had no effect on formyl peptide receptor expression. The results suggest that W-7 primes the fMLP stimulation by direct action on actin leading to breakdown of microfilaments and more expression of formyl peptide receptors, and inhibits the stimulation by indirect action on actin through inactivation of some Ca 2a -dependent proteins resulting in assembly of actin into microfilaments. Which action is favorable depends on the drug concentration. fl 2000 Elsevier Science B.V. All rights reserved.

Increased βcatenin activity correlates with leukemia stem cell expansion and disease progression in chronic myeloid leukemia (CML). We found previously that expression of the CML-related Bcr-abl oncoprotein in myeloid progenitor cells increases expression of Fas-associated phosphatase 1 (Fap1). This resulted in Fap1-dependent resistance to Fas-induced apoptosis in these cells. Fap1 also interacts with the adenomatous polyposis coli (Apc) protein, but the functional significance of this interaction is unknown. Apc participates in a complex that includes glycogen synthase kinase β (Gsk3β) and βcatenin. Assembly of this complex results in phosphorylation of βcatenin by Gsk3β, which facilitates βcatenin ubiquitination and degradation by the proteasome. In this study, we found increased association of Fap1 with the Apc complex in Bcr-abl(+) myeloid progenitor cells. We also found Fap1-dependent inactivation of Gsk3β and consequent stabilization of βcatenin in these cells. Consistent with this, Bcr-abl(+) cells exhibited a Fap1-dependent increase in βcatenin activity. Our studies identified Fap1-dependent Gsk3β inactivation as a molecular mechanism for increased βcatenin activity in CML.

HoxA10 is a homeodomain transcription factor that is involved in maintenance of the myeloid progenitor population and implicated in myeloid leukemogenesis. Previously, we found that FGF2 and CDX4 are direct target genes of HoxA10 and that HOXA10 is a Cdx4 target gene. We also found that increased production of fibroblast growth factor 2 (Fgf2) by HoxA10-overexpressing myeloid progenitor cells results in activation of β-catenin in an autocrine manner. In this study, we identify novel cis elements in the CDX4 and HOXA10 genes that are activated by β-catenin in myeloid progenitor cells. We determine that β-catenin interacts with these cis elements, identifying both CDX4 and HOXA10 as β-catenin target genes in this context. We demonstrate that HoxA10-induced CDX4 transcription is influenced by Fgf2-dependent β-catenin activation. Similarly, Cdx4-induced HOXA10 transcription is influenced by β-catenin in an Fgf2-dependent manner. Increased expression of a set of Hox proteins, including HoxA10, is associated with poor prognosis in acute myeloid leukemia. Cdx4 contributes to leukemogenesis in Hox-overexpressing acute myeloid leukemia, and increased β-catenin activity is also associated with poor prognosis. The current studies identify a molecular mechanisms through which increased expression of HoxA10 increases Cdx4 expression by direct CDX4 activation and by Fgf2-induced β-catenin activity. This results in Cdx4-induced HoxA10-expression, creating a positive feedback mechanism.

HoxA10 is a member of a highly conserved family of homeodomain transcription factors that are involved in definitive hematopoiesis and implicated in the pathogenesis of acute myeloid leukemia (AML). During normal hematopoiesis, HoxA10 facilitates myeloid progenitor expansion and impedes myeloid differentiation. To better understand the molecular mechanisms that control these events, we have been identifying and characterizing HoxA10 target genes. In this study, we identified the gene encoding fibroblast growth factor 2 (Fgf2 or basic fibroblast growth factor) as a target gene that is relevant to the biological effects of HoxA10. We identified two cis elements in the proximal FGF2 promoter that are activated by HoxA10 in myeloid progenitor cells and differentiating phagocytes. We determined that Fgf2 expression and secretion are regulated in a HoxA10-dependent manner in these cells. We found that increased Fgf2 production by HoxA10-overexpressing myeloid progenitor cells induced a phosphoinositol 3-kinase-dependent increase in β-catenin protein. This resulted in autocrine stimulation of proliferation in HoxA10-overexpressing cells and hypersensitivity to other cytokines that share this pathway. Therefore, these studies identified expression of Fgf2 as a mechanism by which HoxA10 controls the size of the myeloid progenitor population. These studies also suggested that aberrant production of Fgf2 may contribute to leukemogenesis in the subset of AML with dysregulated Hox expression. Therapeutic targeting of Fgf2-stimulated signaling pathways might be a rational approach to this poor prognosis subset of AML.

Icsbp is an interferon regulatory transcription factor with leukemia suppressor activity. In previous studies, we identified the gene encoding Fas-associated phosphatase 1 (Fap1; the PTPN13 gene) as an Icsbp target. In the current study, we determine that repression of PTPN13 by Icsbp requires cooperation with Tel and histone deacetylase 3 (Hdac3). These factors form a multiprotein complex that requires pre-binding of Tel to the PTPN13 cis element with subsequent recruitment of Icsbp and Hdac3. We found that knockdown of Tel or Hdac3 in myeloid cells increases Fap1 expression and results in Fap1-dependent resistance to Fas-induced apoptosis. The TEL gene was initially identified due to involvement in leukemia-associated chromosomal translocations. The first identified TEL translocation partner was the gene encoding platelet-derived growth factor receptor β (PdgfRβ). The resulting Tel-PdgfRβ fusion protein exhibits constitutive tyrosine kinase activity and influences cellular proliferation. In the current studies, we find that Tel-PdgfRβ influences apoptosis in a manner that is independent of tyrosine kinase activity. We found that Tel-PdgfRβ expressing myeloid cells have increased Fap1 expression and Fap1-dependent Fas resistance. We determined that interaction between Tel and Tel-PdgfRβ decreases Tel/Icsbp/Hdac3 binding to the PTPN13 cis element, resulting in increased transcription. Therefore, these studies identify a novel mechanism by which the Tel-PdgfRβ oncoprotein may contribute to leukemogenesis.

HoxA10 is a homeodomain transcription factor that influences a number of developmental processes, including hematopoiesis. During definitive hematopoiesis, expression of HoxA10 is maximal in committed myeloid progenitor cells and decreases as differentiation proceeds. Aberrantly increased expression of HoxA10 was found in bone marrow cells in a poor prognosis subset of human acute myeloid leukemia (AML). Consistent with this, AML developed in mice transplanted with HoxA10-overexpressing bone marrow. However, relatively few target genes have been identified that explain the role of HoxA10 in leukemogenesis. In the current study, we identified CDX4 as a HoxA10 target gene. Cdx4 is a homeodomain transcription factor that was also implicated in myeloid leukemogenesis. Although relatively few Cdx4 target genes have been identified, Cdx4 was known to influence HOX gene transcription. We identified a HoxA10-binding cis element in the CDX4 promoter that activated transcription. We also identified a Cdx4-binding cis element that activated the HOXA10 promoter. Therefore, increased Cdx4 expression in HoxA10-overexpressing cells augmented transcription of the endogenous HOXA10 gene. Increased endogenous HoxA10 in these cells induced additional CDX4 transcription. We found that Cdx4 influenced transcription of HoxA10 target genes in a HoxA10-dependent manner. Similarly, HoxA10 influenced transcription of HOX genes in a Cdx4-dependent manner. We previously found that HoxA10-overexpressing myeloid progenitors were hypersensitive to a variety of cytokines. In the current studies, we found that Cdx4 knockdown decreased cytokine hypersensitivity of HoxA10-overexpressing cells. Therefore, these studies identified a positive feedback relationship between HoxA10 and Cdx4, which potentially amplified the contribution of either transcription factor to the pathogenesis of AML.

The homeodomain transcription factor HoxA10 is maximally expressed in myeloid progenitor cells. Sustained HoxA10 expression during differentiation has been described in poor prognosis human acute myeloid leukemia (AML). Consistent with this, engineered overexpression of HoxA10 in murine bone marrow induces a myeloproliferative disorder that progresses to AML over time. This murine model suggests that HoxA10 overexpression is sufficient for myeloproliferation but that differentiation block, and therefore AML, requires acquisition of additional mutations. In myeloid progenitor cells, HoxA10 represses transcription of genes that encode phagocyte effector proteins such as gp91PHOX and p67PHOX. Tyrosine phosphorylation of HoxA10 during myelopoiesis decreases binding to these target genes. In immature myeloid cells, HoxA10 also activates transcription of the DUSP4 gene that encodes Mkp2, an anti-apoptotic protein. HoxA10 binding to the DUSP4 promoter decreases during myelopoiesis. Therefore, both myeloid-specific gene repression and DUSP4 activation by HoxA10 decrease during myelopoiesis. This results in phenotypic differentiation and facilitates apoptosis as differentiation proceeds. HoxA10 is de-phosphorylated by SHP2 protein-tyrosine phosphatase in myeloid progenitors. This mechanism maintains HoxA10 in a nonphosphorylated state in immature, but not differentiating, myeloid cells. Constitutively active SHP2 mutants have been described in human AML, which dephosphorylate HoxA10 throughout myelopoiesis. In this study, we hypothesize that constitutive SHP2 activation synergizes with HoxA10 overexpression to accelerate progression to AML. Because both HoxA10 overexpression and constitutive SHP2 activation are found in poor prognosis human AML, these studies contribute to understanding biochemical aspects of disease progression in myeloid malignancy.

Myeloproliferative disorders (MPDs) are characterized by cytokine hypersensitivity and apoptosis resistance. Development of a block in myeloid differentiation is associated with progression of MPD to acute myeloid leukemia (AML) and portends poor prognosis. Identifying molecular markers of this transition may suggest targets for therapeutic intervention. Interferon consensus sequence binding protein (ICSBP, also known as IRF8) is an interferon-regulatory transcription factor that functions as a leukemia tumor suppressor. In mice, ICSBP deficiency induces an MPD that progresses to AML over time, suggesting that ICSBP deficiency is sufficient for myeloproliferation, but additional genetic lesions are necessary for AML. Since activity of ICSBP is influenced by tyrosine phosphorylation state, we hypothesized that mutations in molecular pathways that regulate this process might synergize with ICSBP deficiency for progression to AML. Consistent with this, we found that constitutive activation of SHP2 protein tyrosine phosphatase synergized with ICSBP haploinsufficiency to facilitate cytokine-induced myeloproliferation, apoptosis resistance, and rapid progression to AML in a murine bone marrow transplantation model. Constitutive SHP2 activation cooperated with ICSBP deficiency to increase the number of progenitors in the bone marrow and myeloid blasts in circulation, indicating a block in differentiation. Since SHP2 activation and ICSBP deficiency may coexist in human myeloid malignancies, our studies have identified a molecular mechanism potentially involved in disease progression in such diseases.

Transcription of the ITGB3 gene, which encodes beta3 integrin, increases during myeloid differentiation. alphavbeta3 integrin mediates adhesion to fibronectin or vitronectin and regulates various aspects of the inflammatory response in mature phagocytes. In these studies, we found that the homeodomain transcription factor HoxA10 interacted with a specific ITGB3 cis element and activated transcription of this gene during myeloid differentiation. We also found that increased fibronectin adhesion in differentiating myeloid cells was dependent upon this HoxA10-induced increase in beta3 integrin expression. We determined that activation of ITGB3 transcription required a HoxA10 domain that was not identical to the "hexapeptide" that mediates interaction of Hox and Pbx proteins. This activation domain was also not identical to a previously identified HoxA10 repression domain that mediates interaction with transcriptional co-repressors. Instead, this HoxA10 activation domain had homology to "PQ" protein-protein interaction domains that have been described previously in other transcription factors. Consistent with this, we found that the HoxA10 PQ-like domain recruited the CREB-binding protein (CBP) to the ITGB3 promoter. This was associated with an increase in local histone acetylation in vivo. In immature myeloid cells, we previously determined that HoxA10 repressed transcription of the CYBB and NCF2 genes, which encode the phagocyte oxidase proteins gp91(PHOX) and p67(PHOX), respectively. Therefore, our studies indicated that HoxA10 either activates or represses gene transcription at various points during myelopoiesis. Our studies also suggested that HoxA10 is a bifunctional protein that is involved in dynamic regulation of multiple aspects of phagocyte phenotype and function.

The CYBB gene encodes gp91Phox; a component of the phagocyte respiratory burst oxidase. CYBB transcription is restricted to myeloid cells differentiated beyond the promyelocyte stage. In undifferentiated myeloid cells, the homeodomain (HD) transcription factor HoxA10 represses CYBB transcription via a cis element in the proximal promoter. During myelopoiesis, phosphorylation of conserved tyrosine residues in the HD decreases HoxA10 binding to this CYBB cis element. In the current studies, we found HoxA9 activates CYBB transcription in differentiated myeloid cells via the same cis element. We find HoxA9-mediated CYBB-transcription requires Pbx1 but is inhibited by Meis1. Additionally, phosphorylation of the conserved HD tyrosines increases HoxA9 binding to the CYBB promoter. The HOXA9 gene is involved in leukemia-associated translocations with the gene encoding Nup98, a nucleopore protein. We find expression of a Nup98-hoxA9 fusion protein blocks HoxA9-induced CYBB transcription in differentiating myeloid cells. In comparison to HoxA9, Nup98-hoxA9 has greater binding affinity for the CYBB cis element, but binding is not altered by HD tyrosine phosphorylation. Therefore, these studies identify CYBB as a common target gene repressed by HoxA10 and activated by HoxA9. These studies also suggest overexpression of Meis1 or Nup98-hoxA9 represses myeloid-specific gene transcription, thereby contributing to differentiation block in leukemogenesis.

The pituitary homeobox factor-1 (Pitx-1) transactivates a number of pituitary-specific genes through direct interaction with other specific transcription factors. We demonstrate here that Pitx-1 plays a crucial role in the regulation of the Chinook salmon luteinizing hormone beta gene promoter through a number of novel mechanisms. On the proximal promoter its action involves a synergistic effect with steroidogenic factor-1 (SF-1) alone or in combination with the estrogen receptor; promoter activity being induced by 9- or 35-fold over controls, respectively. Further upstream, a series of four Pitx-1 response elements (located between 1366 and 1506 bp from the transcriptional start site) is also involved in regulating the promoter activity. The two distal sequences have the greatest effect on the basal activity and are also essential for the gonadotropin-releasing hormone (GnRH) response. Mammalian two-hybrid assays revealed that Pitx-1 can homodimerize. Moreover, circular permutation assays indicate that binding of Pitx-1 to more than one response element induces conformational changes of the target DNA. This constitutes an additional mechanism through which Pitx-1 can mediate transactivation of this gene, allowing the demonstrated interaction of proximal response elements and distal enhancers, thus facilitating the maximal GnRH response that was seen in the longer promoter constructs. Our research also indicates that Pitx-1 is phosphorylated on three residues when bound to the DNA.

Leukemias with chromosomal translocation or partial tandem duplications involving the MLL (mixed lineage leukemia) gene have exceptionally poor prognosis (referred to as 11q23-leukemias). At the molecular level, 11q23-leukemias are characterized by aberrant expression of a set of homeodomain transcription factors in hematopoietic stem cells (HSC) and differentiating myeloid progenitor cells. This transcription factor set includes HoxB3, B4, A7-11, Cdx2-4 and Meis1. Cdx and Hox proteins are involved in regulating hematopoiesis. Transcription of HOX and CDX genes decreases normal myelopoiesis, but is aberrantly sustained in 11q23-leukemias. Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates CDX4 transcription. The events that break this feedback loop, permitting a decrease in Cdx4-expression during normal myelopoiesis, were previously undefined. In the current study, we find that HoxA9 represses CDX4 transcription in differentiating myeloid cells; antagonizing activation by HoxA10. We determine that tyrosine phosphorylation of HoxA10 impairs transcriptional activation of CDX4, but tyrosine phosphorylation of HoxA9 facilitates repression of this gene. Since HoxA9 and HoxA10 are phosphorylated during myelopoiesis, this provides a mechanism for differentiation-stage-specific Cdx4 expression. HoxA9 and HoxA10 are increased in cells expressing Mll-Ell, a leukemia associated MLL1 fusion protein. We find that Mll-Ell induces a HoxA10-dependent increase in Cdx4-expression in myeloid progenitor cells. However, expression of Cdx4 decreases in a HoxA9-dependent manner upon exposure of Mll-Ell-expressing cells to differentiating cytokines. Leukemia associated, constitutively active mutants of Shp2 block cytokine-induced tyrosine phosphorylation of HoxA9 and HoxA10. In comparison to cells expressing Mll-Ell alone, we find that co-expression of Mll-Ell plus constitutively active Shp2 increases CDX4 transcription and Cdx4 expression in myeloid progenitor cells. And, increased Cdx4 expression is sustained upon exposure of these cells to differentiating cytokines. Our results identify a mechanism for increased and sustained CDX4 transcription in leukemias co-overexpressing HoxA9 and HoxA10 in combination with constitutive activation of Shp2. We also demonstrate that inhibition of Shp2-PTP activity decreases Cdx4 expression in Hox-overexpressing human myeloid leukemias. Disclosures No relevant conflicts of interest to declare.

Leukemias with chromosomal translocation or partial tandem duplications involving the MLL (mixed lineage leukemia) gene have exceptionally poor prognosis (referred to as 11q23-leukemias). At the molecular level, 11q23-leukemias are characterized by increased expression of a set of homeodomain transcription factors in CD34+ hematopoietic stem cells (HSC) and differentiating CD34- myeloid progenitor cells. This transcription factor set includes HoxB3, B4, A7-11 and Meis1. Although many elegant studies have identified the precise molecular mechanisms by which MLL-fusion proteins increase HOX gene transcription, less is known about the downstream effects of increased Hox expression and potential implications for leukemogenesis. In prior studies, we determined that the gene encoding fibroblast growth factor 2 (Fgf2 or basic Fgf) is a HoxA10 target gene, and production of Fgf2 is increased in HoxA10 overexpressing primary bone marrow cells. In the current studies, we determine that FGF2 is also activated by HoxA9; identifying the only common target gene for HoxA9 and HoxA10 where both proteins activate transcription. We also find Mll-Ell (an MLL-fusion protein) increases FGF2 transcription and Fgf2 expression in a HoxA9 and HoxA10 dependent manner. Importantly, autocrine production of Fgf2 by Mll-Ell expressing HSC and myeloid progenitor cells increases cytokine-induced proliferation and enhances cell survival. In additional studies, we determine that these effects of Fgf2 are mediated by phospho inositol 3 kinase (PI3K) dependent stabilization of beta catenin. We find that autocrine production of Fgf2 results in increased expression of key beta catenin target genes in Mll-Ell expressing cells, including c-myc, survivin and cyclinD1. Beta catenin target genes also include the genes encoding CDX4 and HOXA10. Additionally, Cdx4 activates transcription of the HOXA9 and HOXA10 genes, and HoxA10 activates the CDX4 gene. Therefore, increased Fgf2 also establishes a positive feedback loop that enhances transcriptional activation of HOXA9 and HOXA10 by MLL-fusion proteins. 11q23-AML is generally refractory, but the molecular mechanisms for this are unknown. Our studies suggest that increased Fgf2 expression in 11q23-AML contributes to cytokine hypersensitivity and sustained survival of leukemia stem cells. Targeting the effects of Fgf2 might increase chemotherapy sensitivity in this form of leukemia. Small molecule inhibitors of Fgf-receptors are available, but have not yet been tested in this form of AML. We suggest that this would be a rational therapeutic approach to this disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 2404 Fap1 (Fas-associated phosphatase 1) interacts with the C-terminus of multiple proteins via a PDZ-type protein-protein interaction domain. Interaction of Fap1 with Fas results in Fas-de-phosphorylation and inhibits Fas-induced apoptosis. In previous studies, we determined that PTPN13, the gene encoding Fap1, is a target gene for the interferon consensus sequence binding protein (referred to as Icsbp or Irf8). Icsbp functions as a leukemia suppressor in chronic myeloid leukemia (CML), and we previously demonstrated Icsbp/Fap1-dependent apoptosis resistance in cells expressing the CML-associated oncoprotein, Bcr-abl. Another known interaction partner for Fap1 is the adenomatous polyposis coli protein (Apc). However, the functional significance of this interaction has not been previously explored. In the current stuides, we investigate whether interaction between Fap1 and Apc impacts the pathogenesis of CML. Apc is involved in assembly of a multi-protein complex that includes Apc, Axin, Gsk3-beta and beta-catenin. Association of these proteins results in serine/threonine phosphorylation (pS/T) of beta-catenin by Gsk3-beta. pS/T beta-catenin is released from the complex and degraded by the proteasome. Previous studies demonstrated that pS/T of beta-catenin, and consequent beta-catenin degradation, is impaired in CML, but the mechanism for this impairment is unknown in most cases. We find that Icsbp-dependent increase in Fap1 results in increased interaction between Fap1 and Apc in Bcr-abl+ myeloid progenitor cells. We also find that increased Fap1/Apc interaction is associated with inactivation (de-phosphorylation) of Gsk3-beta and a decrease in inhibitory pS/T of βcatenin. We find that this results in increased beta-catenin protein and activity. We also find that Fap1 co-immuno-precipitates with Apc, Axin, Gsk3-beta and beta-catenin. This result suggests the possibility that Gsk3-beta is a substrate for Fap1 protein tyrosine phosphatase activity. Consistent with this hypothesis, we find that Fap1 is able to dephosphorylate Gsk3-beta in vitro. We tested the functional significance of this observation in experiments using an activated form of Gsk3-beta that cannot be dephosphorylated at a key residue (Y216D-Gsk3-beta). We find that expression of this Gsk3-beta mutant partly reverses the effect of Bcr-abl and Fap1 on beta-catenin protein stability and activity. Increased beta-catenin activity is a characteristic of leukemia stem cells (LSC) in CML, and increasing beta-catenin activity in the bone marrow is associated with poor prognosis in CML. Therefore, these studies identify a novel pathway that regulates events significant to CML pathogenesis. These studies also identify a role for Apc in the pathogenesis of leukemia, and another mechanism for the leukemia suppressor effect of Icsbp. Disclosures: No relevant conflicts of interest to declare.

HOX genes encode highly conserved homeodomain (HD) transcription factors and are arranged in four groups (A–D). During definitive hematopoiesis, HOX gene expression is activated 3′ to 5′ through each group. Therefore, HOX1-4 are actively transcribed in hematopoietic stem cells and HOX7-11 in committed progenitors. Under normal conditions, HoxA7-11 expression decreases during CD34+ to CD34− maturation. Abnormal Hox expression is characteristic of several poor prognosis subtypes of Acute Myeloid Leukemia (AML) including AML with translocations or duplications of the MLL gene. In such leukemias, expression of HoxB3, B4 and A7-11 is sustained in CD34−CD38+ cells. In murine bone marrow transplantation experiments, expression of MLL fusion proteins, HoxA9 or HoxA10 induces a myeloproliferative disorder (MPD) characterized by increased neutrophils (PMN). Over time, the mice progress to AML with circulating myeloid blasts. These results suggest overexpression of HoxA9 or HoxA10 is adequate for MPD, but differentiation block (AML) requires additional lesions. We found that HoxA9 and HoxA10 proteins not only decrease in expression during the CD34+ to CD34− transition, but also are tyrosine phosphorylated. In additional studies, we found that HoxA10 tyrosine phosphorylation state is relevant for differentiation stage-specific target gene expression during myelopoiesis. HoxA10 represses genes encoding phagocyte effector proteins in undifferentiated myeloid cells. During myelopoiesis, phosphorylation of conserved HD-HoxA10 tyrosines decreases binding to these genes, permitting phenotypic and functional differentiation. HoxA10 activates transcription of the gene encoding Mkp2 (Dusp4) in myeloid progenitors. Decrease in HoxA10-binding to this gene as differentiation proceeds decreases transcription and renders the cells susceptible to Jnk induced apoptosis. Therefore, we hypothesized that genetic lesions which influence post translational modification might cooperate with HoxA10 overexpression to lead from MPD to AML. In myeloid progenitors, HoxA10 is maintained in a non-phosphorylated state by SHP2 protein tyrosine phosphatase. SHP2 activity decreases as differentiation proceeds. Activating mutations in SHP2 have been described in AML. We found that such activated SHP2 mutants dephosphorylate HoxA10 through out ex vivo myelopoiesis. Therefore, we investigated cooperation between these two leukemia associated abnormalities in vivo. Mice were transplanted with bone marrow overexpressing HoxA10 (or empty vector control) with or without activated SHP2 (E76K). To control for SHP2 overexpression, other mice were transplanted with bone marrow overexpressing HoxA10 and wild type SHP2. Mice transplanted with bone marrow overexpressing HoxA10 (±SHP2) developed MPD which evolved to AML over 4 mos, consistent with previous observations. However, mice transplanted with bone marrow overexpressing HoxA10 and E76K SHP2 developed AML within 4 wks. This rapid development of AML correlated with abnormalities in expression of myeloid specific HoxA10 target genes. These studies indicate the importance of HoxA10 post translational modification for physiologically relevant function and identify cooperating lesions which may be significant for disease progression in human AML.

Transcription of the CYBB gene, which encodes the respiratory burst oxidase protein, gp91phox, is restricted to phagocytic cells, differentiated beyond the promyelocyte stage. Regulation of CYBB transcription involves several repressor cis elements in the promoter. These repressor elements are homologous to the derived consensus sequence for DNA-binding of HoxA10 as a hetero-dimer with Pbx1a. The HoxA10/Pbx1 complex recruits histone deacetylase 2 and represses CYBB transcription in undifferentiated cells. During myeloid differentiation, HoxA10 is tyrosine phosphorylated, which decreases DNA-binding affinity for the CYBB repressor element. In our previous investigations, we found this decreased binding affinity requires phosphorylation of two tyrosine residues in the DNA-binding homeodomain. These residues are highly conserved with other HoxA proteins. However, we also found decreased DNA-binding requires interaction of these homeodomain tyrosines with a non-conserved domain, amino terminal in HoxA10. In previous investigations, we determined these CYBB repressor cis elements function as positive cis elements in differentiated myeloid cells, and interact with an unidentified protein complex. In these studies, we investigate the proteins involved in transcriptional activation of the CYBB gene, via these promoter sequences. We find this cis element is activated by HoxA9 and Pbx1a, in differentiating myeloid cells. Consistent with this, we find increased binding of HoxA9 to this CYBB cis element, during myeloid differentiation, in vitro and in vivo. Consistent with our results with HoxA10, we find HoxA9 is tyrosine phosphorylated during myeloid differentiation. However, in contrast to HoxA10, we find tyrosine phosphorylation of the conserved residues in the HoxA9 DNA-binding homeodomain increases binding to the CYBB cis element. Therefore, we conclude HoxA10 and HoxA9 have opposing functions, for myeloid specific gene transcription, during differentiation. We also find these functions are regulated by post translational modification of conserved tyrosine residues in the homeodomain regions of these proteins. Additionally, our results suggest differences in the effect of phosphorylation of these residues on DNA-binding is likely due to interaction with non-conserved domains in HoxA9 and HoxA10.