Background: Amyloid β (Aβ)-directed immunotherapy has shown promising results in preclinical and early clinical Alzheimer’s disease (AD) trials, but successful translation to late clinics has failed so far. Compelling evidence suggests that post-translationally modified Aβ peptides might play a decisive role in onset and progression of AD and first clinical trials targeting such Aβ variants have been initiated. Modified Aβ represents a small fraction of deposited material in plaques compared to pan-Aβ epitopes, opening up pathways for tailored approaches of immunotherapy. Here, we generated the first monoclonal antibodies that recognize L-isoaspartate-modified Aβ (isoD7-Aβ) and tested a lead antibody molecule in 5xFAD mice. Methods: This work comprises a combination of chemical and biochemical techniques as well as behavioral analyses. Aβ peptides, containing L-isoaspartate at position 7, were chemically synthesized and used for immunization of mice and antibody screening methods. Biochemical methods included anti-isoD7-Aβ monoclonal antibody characterization by surface plasmon resonance, immunohistochemical staining of human and transgenic mouse brain, and the development and application of isoD7-Aβ ELISA as well as different non-modified Aβ ELISA. For antibody treatment studies, 12 mg/kg anti-isoD7-Aβ antibody K11_IgG2a was applied intraperitoneally to 5xFAD mice for 38 weeks. Treatment controls implemented were IgG2a isotype as negative and 3D6_IgG2a, the parent molecule of bapineuzumab, as positive control antibodies. Behavioral studies included elevated plus maze, pole test, and Morris water maze. Results: Our advanced antibody K11 showed a KD in the low nM range and > 400fold selectivity for isoD7-Aβ compared to other Aβ variants. By using this antibody, we demonstrated that formation of isoD7-Aβ may occur after formation of aggregates; hence, the presence of the isoD7-modification differentiates aged Aβ from newly formed peptides. Importantly, we also show that the Tottori mutation responsible for early-onset AD in a Japanese pedigree is characterized by massively accelerated formation of isoD7-Aβ in cell culture. The presence of isoD7-Aβ was verified by K11 in post mortem human cortex and 5xFAD mouse brain tissue. Passive immunization of 5xFAD mice resulted in a significant reduction of isoD7-Aβ and total Aβ in brain. Amelioration of cognitive impairment was demonstrated by Morris water maze, elevated plus maze, pole, and contextual fear conditioning tests. Interestingly, despite the lower abundance of the isoD7-Aβ epitope, the application of anti-isoD7-Aβ antibodies showed comparable treatment efficacy in terms of reduction of brain amyloid and spatial learning but did not result in an increase of plasma Aβ concentration as observed with 3D6 treatment. Conclusions: The present study demonstrates, for the first time, that the antibody-mediated targeting of isoD7- modified Aβ peptides leads to attenuation of AD-like amyloid pathology. In conjunction with previously published data on antibodies directed against pGlu-modified Aβ, the results highlight the crucial role of modified Aβ peptides in AD pathophysiology. Hence, the results also underscore the therapeutic potential of targeting modified amyloid species for defining tailored approaches in AD therapy.

Fungal infections are one of the leading causes of death in humans, causing infections that range from mild superficial infections to severe, and life-threatening invasive infections that affect the bloodstream and vital organs. Invasive fungal infections have a high mortality rate, leading to approximately 1.5 million deaths annually. The most common pathogens responsible for these infections are Candida, Cryptococcus, and Aspergillus. Currently, treatment options for invasive fungal infections are limited to three main classes of antifungal drugs: azoles, polyenes, and echinocandins. The emergence of new fungal species, such as Candida auris, which displays high resistance and mortality rates (30-60%), has further complicated treatment efforts. Thus, there is a critical need for new therapeutic strategies to combat these life-threatening pathogens. C. auris isolates have demonstrated significant resistance, especially to azoles (fluconazole) and polyenes (amphotericin B, AmB). To address this, we screened approximately 2,600 FDA-approved drugs and clinical compounds to identify agents capable of inhibiting C. auris growth and enhancing or restoring the antifungal activity of existing antifungals. This screening revealed that HIV protease inhibitors, such as lopinavir, atazanavir, saquinavir and ritonavir, significantly enhanced the antifungal activity of azoles (fluconazole, voriconazole, itraconazole, and posaconazole) and polyene (AmB). Mechanistic studies showed that the HIV protease inhibitors inhibited the fungal efflux pump, and interfered with glucose utilization, leading to reduced ATP levels in C. auris. Moreover, HIV protease inhibitors, in combination with AmB, were able to inhibit the virulence factors of Candida species. Furthermore, HIV protease inhibitors, in combination with itraconazole or posaconazole, resulted in a significant reduction of the C. auris burden in mice kidneys. Two additional drugs, darapladib and rilapladib, phospholipase A2 inhibitors, were identified as potent inhibitors of C. auris. Darapladib and rilapladib demonstrated superior killing kinetics compared to itraconazole. Interestingly, C. auris did not develop any detectable resistance to both drugs at sub-inhibitory concentration over 16-passages. Mechanistic studies revealed that darapladib increased the plasma membrane permeability and caused DNA leakage, likely due to a direct interaction with ergosterol, as suggested by competition assays with exogenous ergosterol. We further validated the broad-spectrum, fungicidal, and potent activity of darapladib in combination with AmB, showing significant synergy against multidrug-resistant fungal pathogens, including C. auris, Aspergillus fumigatus and mucormycosis-associated species. Additionally, darapladib demonstrated a superior safety profile compared to AmB, exhibiting lower affinity for cholesterol, and no toxicity in combination with AmB to kidney cells. Finally, rilapladib (at 2× MIC) inhibited the C. auris burden by 1.5 Log10, whereas darapladib (at 2× MIC) achieved complete eradication of the C. auris burden in an in vivo C. elegans model. / Doctor of Philosophy / Fungi are a group of living organisms that are neither plants nor animals. While Some fungi are beneficial, others can cause serious diseases in humans. Candida auris is a human fungal pathogen that causes serious infections with mortality rate up to 60%. It has become a growing threat to global public health. This pathogen is particularly concerning due to its high virulence, resistance to multiple antifungal drugs, and ability to rapidly spread, often leading to hospital outbreaks. In our current research, we have identified several compounds that either inhibit the growth of C. auris and/or enhance the efficacy of existing antifungals, such as azoles and amphotericin B. We have identified HIV protease inhibitors (lopinavir, atazanavir, saquinavir, and ritonavir) that were able to enhance the antifungal activities of both azoles (fluconazole, itraconazole, voriconazole, and posaconazole) and polyene (amphotericin B) against multidrug-resistant C. auris. The combination of HIV protease inhibitors with amphotericin B inhibited the virulence factors of Candida species, including biofilm and hyphae formation. Two drugs darapladib and rilapladib were found to not only inhibit the growth of C. auris alone but also enhance the antifungal activity of amphotericin B against all medically important fungal pathogens tested. Additionally, we have explored the mechanisms by which these compounds act on the pathogen. Finally, we have confirmed the effectiveness of these drugs in live models, including mice and worms.

Age and sex are risk factors of Alzheimer's disease (AD). Among the neurotransmitter systems, gamma-aminobutyric acid (GABA) has been implicated in AD pathogenesis but the relevance of sex-specific GABAergic dysfunction during AD progression remains unknown. In the present study, we utilized state-of-the-art high-resolution magic angle spinning nuclear magnetic resonance to systematically monitor the brain region-, age-, and sex-specific modulation of GABA levels in wild-type and Tg2576 mice with amyloid pathology. In addition, we followed the possible role of reactive astrocytes in sex-specific GABA modulation. In female Tg2576 mice, hippocampal GABA levels were significantly elevated, along with higher number of reactive astrocytes and amyloid deposition. The elevated GABA was found to be produced via the monoamine oxidase-B route from putrescine in reactive astrocytes, more substantially in female than male mice, thus suggesting a role of astrocytes in memory impairment and sex-related differences in AD. Our results paint a coherent model of memory impairment in AD and signify that dynamic changes in regional GABA may be at the root of marked sex disparities observed in AD.

Alveolar macrophages comprising up to 95% of the pulmonary alveoli, are the gate-keepers of homeostasis by ensuring efficient tissue function through metabolizing excessive surfactant and phagocyting inhaled day-to-day and innocuous pathogens and particles, without triggering an immune response. Despite that, they are capable of orchestrating a very well-balanced immune response upon invasion of pathogens. These embryonic-derived cells are capable of self-renewal and therefore maintain themselves in the lungs throughout adult life, with minimal contribution from the circulating monocytes. This self-renewal capacity is attained intrinsically by maintaining low levels of transcription factors MafB and cMaf, and extrinsically through two main cytokines, namely GM-CSF secreted by alveolar epithelial type II cells, and TGFb secreted by AMs themselves in an autocrine manner. However, in inflammatory conditions such as acute respiratory distress syndrome (ARDS), depletion of AM pool and upregulation of MAFB among lung macrophages have been reported. Keeping in mind the role of transcription factors MafB and cMaf in inhibiting proliferative capacity of macrophages; we hypothesized that this depletion is due to upregulation of MafB and hence the suppression of enhancer regions of self-renewal genes in AMs. To investigate the role of MafB and its compensatory partner cMaf in ARDS, we have established a mouse model of ARDS using oropharyngeal instillation of LPS in WT and MafB/cMaf double-knockout (Maf-DKO) mice. Alongside, the molecular mechanisms of the effect of LPS on AMs was investigated ex-vivo. The obtained results have clearly shown that ex-vivo, LPS inhibits proliferation of AMs in a dose dependent manner, and induces apoptosis significantly. Regain of proliferative potential of LPS-stimulated AMs was evident upon TLR4 inhibition, and MyD-88 was shown to be the dominant adaptor downstream of TLR4 (as opposed to TRIF). Both WT and DKO AMs responded to LPS stimulation within 2 hours, by switching from OXPHOS to glycolysis, which accounts for their efficient pro-inflammatory phenotype once activated. Upon activation, MafB and cMaf were upregulated after 48 hours and the inhibition of AM proliferation was shown to be Maf-independent. Similarly, depletion of AM pool was shown to be Maf independent invivo, evident by similar kinetics of AM numbers in WT and DKO at different timepoints upon LPS stimulation. However, several findings indicated potential advantage of Maf-deficiency in tissue regeneration; this includes: 1) higher number of Ly6C+ monocytes and their earlier differentiation into resident AMs, 2) lower degree of tissue damage revealed by H&E staining, 3) higher number of alveolar epithelial type II cells, 4) significantly higher levels of cytotoxicity pointing towards cellular turnover, and 5) significantly higher levels of SP-D and thus its antiinflammatory effects. In a quest for investigating factors which could enhance proliferative potential of AMs and ultimately neutralize the inhibitory effect of LPS, the impact of TGFb and ActivinA was studied. I have shown that TGFb and to a higher extend ActivinA boost the proliferation rate of AMs, ex-vivo. The autocrine effect of these cytokines was validated by blocking signal transduction through inhibition of SMAD2/3, which resulted in a significant increase in doubling time of AMs. Interestingly, Inhba was shown to be significantly upregulated in AMs, as opposed to TGFb. The importance of ActivinA was further demonstrated by its direct inhibition and the resulting reduction in growth rate of AMs. On the contrary to the significant role of these cytokines in enhancing the growth rate of AMs ex-vivo, they could rescue AM proliferation under the effect of LPS. In conclusion, I have demonstrated that LPS inhibits AM proliferation in a dose dependent and Maf-independent manner. Furthermore, neither TGFb nor ActivinA could rescue proliferation of LPS-stimulated AMs. Although Maf-deficiency was not shown to be beneficial during the inflammatory phase of ARDS, due to the fact that both WT and DKO AMs were equally depleted at the peak of inflammation, multiple data indicated potential advantage of Maf deficiency during resolution phase.

No / The interleukin-2-deficient (IL-2¿/¿) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2¿/¿ mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2¿/¿ mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-B (p65) activation with the inhibitory p50 form of NF-B predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.

Die schlechte Prognose der meisten disseminierten Tumorerkrankungen ist häufig in einer vorbestehenden oder erworbenen Resistenz gegenüber Zytostatika begründet. Da die meisten Zytostatika mit zellulären Strukturen interagieren, war lange angenommen worden, dass der antineoplastische Effekt unmittelbar durch massive Zellschädigung bewirkt wird. Hieraus folgte, dass Chemoresistenz auf Mechanismen beruhen müsse, welche das Zytostatikum an der Wechselwirkung mit seiner intrazellulären Zielstruktur hindern. Arbeiten der letzten Jahre haben jedoch gezeigt, dass die meisten Zytostatika indirekt über DNA-Schädigung ein relativ uniformes, genetisch kodiertes Zelltod-Programm auslösen, demzufolge postuliert wurde, dass auch Apoptosedefekte für "Multi-Drug-Resistenz" verantwortlich sein könnten. Allerdings ist der tatsächlich Beitrag zytostatika-induzierter Apoptose am Therapieerfolg nicht geklärt, wobei der Wahl geeigneter Testsysteme eine wesentliche Bedeutung für diese Kontroverse zuzukommen scheint. Gegenstand der vorliegenden Arbeit ist daher die Etablierung eines transgenen Lymphom-Modells, in welchem chemotherapeutische Effekte an spontan entstandenen Tumoren mit definierten genetischen Läsionen in ihrer natürlichen Umgebung untersucht werden können. Hierbei konnte gezeigt werden, dass Mutationen in apoptose-relevanten Genloci wie p53, INK4a/ARF oder bcl2 sowohl die Manifestation myc-transgener Lymphome dramatisch beschleunigen, als auch den Therapieerfolg kompromittieren. Neben Apoptose wurde darüberhinaus prämature Seneszenz, ein terminaler Zellzyklus-Arrest, als prognose-relevantes Chemotherapie-Effektorprogramm identifiziert. Damit dokumentiert die vorliegende Arbeit einen wichtigen Zusammenhang von Gendefekten, die während der Tumorigenese erworben wurden, und später evidenter Chemoresistenz, wobei manche Mutationen bereits vor Zytostatika-Exposition Resistenz begründen können. Die Identifikation und pharmakogenomische Charakterisierung potentiell resistenz-vermittelnder Gene und Mutationen in relevanten Testsystemen wird für die Entwicklung spezifischerer, aber weniger toxischer "targeted Therapeutics" von großer Bedeutung sein. / Intrinsic or acquired chemoresistance is the major cause for the adverse outcome of disseminated malignancies. The fact that most anticancer agents bind to subcellular targets prompted the assumption that drug-induced cytotoxicity must be a direct consequence of severe cellular damage. Hence, chemoresistance was thought to arise from mechanisms that prevent or disrupt the drug-target interaction. By contrast, more recent data suggested that DNA damage caused by most, if not all, anticancer agents may trigger a relatively uniform, genetically encoded cell death program. In turn, defects in the apoptotic machinery should account for multi-drug resistance as well. However, due to technical limitations of current test systems, it has been difficult to assess the overall contribution of apoptotic cell death to treatment outcome. In the studies presented here, a transgenic mouse lymphoma model was established in order to exploit drug responses of spontaneously developed malignancies growing at their natural sites but harboring defined genetic defects. Using this model, alterations in apoptosis-related gene loci such as p53, INK4a/ARF or bcl2 result in both dramatic acceleration of myc-driven lymphomagenesis and compromised treatment responses. Importantly, not only apoptosis, but premature senescence, a terminal cell-cycle arrest, was found to impact on treatment outcome. In essence, this work describes and important connection between cancer genes and cancer therapy, i.e. genetic defects acquired during tumorigenesis may already co-select for chemoresistance prior to any drug encounter. The identification and pharmacogenomic evaluation of resistance conferring candidate genes and mutations using adequate test systems is likely to play a key role in the development of novel, more specific but less toxic so called "targeted Therapeutics".

Im Zentrum der vorliegenden Arbeit steht die Proteomanalyse des APP23-Mausmodells für die Alzheimer-Krankheit (AK). Es wurden die Gehirnregionen Hippocampus und Cortex der Altersstadien Embryonaltag 16 sowie 1, 2, 7 und 15 Monate untersucht und somit erstmals eine Art „Proteom-Lebensprofil“ eines Mausmodells erstellt. Bei dem Vergleich der APP23-Mäuse mit Wildtypmäusen konnte innnerhalb aller untersuchten Altersstadien und Gehirnregionen eine große Anzahl quantitativer Proteinexpressionsunterschiede festgestellt werden. Interessanterweise bestand jedoch im Hippocampus adoleszenter, zwei Monate alter Mäuse, ein herausragend großer Unterschied. Ein Vergleich der Proteomzusammensetzung zwischen den verschiedenen Altersstadien zeigte, dass spezifisch im Hippocampus der adoleszenten APP23-Mäuse viele entwicklungsbedingte Proteomveränderungen ausgeblieben waren. Zusammen mit der Beobachtung, dass in diesem Altersstadium viele synaptische Proteine in den APP23-Mäusen herunterreguliert waren, weisen die gewonnenen Daten darauf hin, dass ein natürlicher Peak in der hippocampalen Plastizität während der Adoleszenzphase in den APP23-Mäusen ausgeblieben war. Es ist weiterhin bekannt, dass APP als auch eine wichtige Rolle in der embryonalen Neurogenese spielt. Da über diese Prozesse noch wenig bekannt ist, wurde im Rahmen der vorliegenden Studie weiterhin eine grundlegende Untersuchung der murinen embryonalen Gehirnentwicklung durchgeführt. Dabei wurde beobachtet, dass innerhalb eines Zeitraums von zwei Entwicklungstagen die Anzahl an quantitativ veränderten Proteinen konstant ist. Dies könnte die maximal mögliche Veränderungsrate wiederspiegeln, die wiederum einen begrenzenden Faktor für die Geschwindigkeit der Embryonalentwicklung darstellt. Weiterhin stieg im Zuge der Neurogenese die Konzentration zelltypspezifischer Proteine an während im Gegenzug die Konzentration unspezifischer Proteine abnahm. / The work presented here focuses on the analysis of the APP23 mouse model for alzheimer´s disease (AK). Using a proteomics approach; the two brain regions cortex and hippocampus, were analyzed at the ages 1,2,7 and 15 months and at embryonic day 16. Thus, for the first time, a lifetime profile of brain proteome alterations caused by mutant APP expression was created. Protein expression alterations between APP23 and control mice were numerous at all age stages but unexpectedly, the hippocampus of two-month old (adolescent) mice, showed a dramatic peak in the number of altered proteins. When comparing proteome patterns longitudinally between age-stages, protein alterations were largely absent in the hippocampus of adolescent APP23-mice but not in other stages compared. Apparently, the large difference in hippocampal protein pattern changes between two-month old APP23- and wildtype-mice was caused by an absence of distinct developmental changes in APP23-mice. In summary, the absence of longitudinal developmental proteome alterations during adolescence, a developmental stage where neuronal plasticity is prominent and horizontal (disease/control) down-regulation of many proteins related to plasticity suggests the disruption of a normally occurring peak of hippocampal plasticity during adolescence of APP23-mice. APP was also suggested to play an important role in embryonic neurogenesis. Since information about the molecular mechanisms of neurogenesis is scarce, a basic analysis of protein changes during normal embryonic neurogenesis was conducted. Interestingly, the rate of protein concentration change within two days of development was constant. This might represent the maximal rate limiting the speed of embryonic development. During neurogenesis, cell-type specific proteins were up- and unspecific proteins down-regulated. In summary, this study shows that it is of utmost importance to investigate AK even at an early age prior to the occurrence of disease symptoms.

The wide scale use of ZnO nanoparticles (NPs) in the world consumer market has resulted in likelihood of exposure to human beings. The present study was aimed to assess the in vitro and in vivo interactions of ZnO NPs in the mammalian system and to elucidate the possible mechanism of their toxicity. Our in vitro results using human epidermal cells (A431), primary human epidermal keratinocytes and human liver cells (HepG2) demonstrated that cells exposed to ZnO NPs exhibit a decrease in cell viability which was independent of NP dissolution. ZnO NPs also induced oxidative DNA damage as evidenced by an increase in the Fpg sensitive sites. The reactive oxygen species triggered a decrease in mitochondrial membrane potential and an increase in the ratio of Bax/Bcl2 leading to apoptosis through the intrinsic pathway. In addition, ZnO NPs induced phosphorylation of JNK, P38 and P53ser15. The results from our in vivo studies using a mouse model showed that ZnO NPs induce lipid peroxidation, oxidative DNA damage and apoptosis in liver which further confirmed our in vitro findings. The data from the present study provide valuable insights into the cellular interactions of ZnO NPs and the underlying molecular mechanism of their toxicity. The results also stress the need for a comprehensive environmental health and safety assessment of engineered nanomaterials to ensure safer nanotechnology based products.

La protéine de fusion E2A-PBX1 induit une leucémie lymphoblastique aigüe des cellules B pédiatrique chez l’humain. E2A-PBX1 possède de puissantes propriétés de trans-activation et peut se lier à l’ADN ainsi qu’aux protéines homéotiques (HOX) via des domaines conservés dans sa portion PBX1, ce qui suggère qu’une dérégulation des gènes cibles de HOX/PBX1 contribue à la leucémogénèse. Précédemment, Bijl et al. (2008) ont démontré que certains gènes Hox collaborent de manière oncogénique avec E2A-PBX1, et que ces interactions sont cellules-spécifiques et varient en fonction du gène Hox impliqué. Une mutagénèse d’insertion provirale suggère et supporte la collaboration des gènes Hoxa et E2A-PBX1 lors de la leucémogénèse des cellules B. La présence de ces interactions dans les cellules B et leur implication dans l’induction des B-ALL est pertinente pour la compréhension de la maladie humaine, et reste encore mal comprise. Notre étude démontre qu’Hoxa9 confère un avantage prolifératif aux cellules B E2A-PBX1. Des expériences de transplantation à l’aide de cellules B E2A-PBX1/Hoxa9 positives isolées de chimères de moelle osseuse démontrent qu’Hoxa9 collabore avec E2A-PBX1 en contribuant à la transformation oncogénique des cellules, et qu’Hoxa9 seul n’induit aucune transformation. Une analyse par Q-RT-PCR nous a permis de démontrer une forte inhibition de gènes spécifiques aux cellules B dans les leucémies co-exprimant Hoxa9 et E2A-PBX1, en plus d’une activation de Flt3, suggérant une inhibition de la différenciation des cellules B accompagnée d’une augmentation de la prolifération. De plus, la surexpression de Hoxa9 dans des cellules leucémiques de souris transgéniques E2A-PBX1, confère aussi un avantage prolifératif aux cellules in vitro, qui semblent être influencé par une augmentation de l’expression de Flt3 et Pdgfδ. En conclusion, nous démontrons pour la première fois à l’aide d’un modèle murin qu’Hoxa9 collabore avec E2A-PBX1 lors de la transformation oncogénique des cellules B et que la signalisation via Flt3 est impliquée, ce qui est potentiellement pertinent pour la maladie humaine. / The fusion protein E2A-PBX1 induces pediatric B cell leukemia in human. It has strong transactivating properties and can bind to DNA and homeobox (HOX) proteins through conserved domains in the PBX1 portion, suggesting that deregulation of HOX/PBX target genes contribute to leukemogenesis. Previously, we reported oncogenic interactions between Hox genes and E2A-PBX1, which are dependent on cell type as well as on the particular Hox member. A proviral insertional mutagenesis screen provided support for collaboration between Hoxa genes and E2A-PBX1 in B cell leukemogenesis. Whether these interactions occur in B cells and lead to B-ALL, relevant for human disease is still not clear. Here we report that Hoxa9 confers a proliferative advantage to E2A-PBX1 B cells. Transplantation experiments with E2APBX1/Hoxa9 positive B cells isolated from bone marrow (BM) chimeras showed that Hoxa9 interacts with E2A-PBX1 contributing to the oncogenic transformation of B cells, but is unable to transform B cells alone. Q-RT-PCR analysis demonstrated a strong repression of B cell specific genes in leukemias co-overexpressing Hoxa9 and E2A-PBX1 in addition to Flt3 activation, indicating inhibition of B cell differentiation in combination with enhanced proliferation. Overexpression of Hoxa9 in E2A-PBX1 mouse leukemic B cells also resulted in a growth advantage in vitro, likely mediated by the enhanced expression of Flt3 and Pdgfδ. In conclusion we show for the first time that Hoxa9 collaborates with E2A-PBX1 in the oncogenic transformation of B cells in a mouse model that involves Flt3 signaling, which is potentially relevant to human disease.

Increasingly the role of epigenetic machinery as a bridge between underlying DNA sequence and cellular phenotype is being discovered. The establishment of a myriad of unique cellular types sharing identical gene sequences in a multicellular organism gives a broad sense for the inherent role of epigenetic influence on cell differentiation. Importantly, the epigenetic mechanisms involved in establishing cell identity unsurprisingly contribute to diseased states, including cancer. Recent research continues to elucidate contributory roles of epigenetic mechanisms, such as DNA methylation, histone modification, and microRNA regulation, in human cancers. Additionally, chromatin remodelers, such as the Nucleosome Remodeling Factor (NURF), have been identified as important regulators for normal cell biology. While much has been done to identify and characterize the role of NURF chromatin remodeling complex as a key regulator of development in a number of model organisms, little has been published on the implications of NURF in diseases such as cancer. Our preliminary data shows dysregulation of E-cadherins, N-cadherins, and MHC-I genes in Bptf (an essential subunit of NURF) knocked down murine breast cancer cell lines. These proteins have well documented roles in the development and metastatic progression of cancers. To study the effect of Bptf knockdown on the development and progression of cancer we injected Bptf knocked down mouse breast cancer cell lines, 4T1, 66cl4, and 67NR, into syngenic BALB/c mice. Our findings reveal decreased tumor growth in 66cl4 and 67NR as measured by tumor weight at 3-4 weeks post injection. Tumor growth did not appear to be significantly affected in 4T1 challenged mice. However, mice inoculated with Bptf knockdown 4T1 cell lines have decreased metastasis to lungs as compared to control while metastasis of 66cl4 tumors to the lungs appear unaffected. To assess the role of the immune system in decreasing tumor growth in BALB/c mice, we injected 66cl4 tumors into NOD-SCID-Gamma (NSG) immune deficient mice. The tumors from these mice show no difference in tumor growth between Bptf knockdown and control tumors, implicating a role for the immune system regulating the decreased tumor weight in BALB/c mice. To delineate which immune cell effector may impede breast cancer carcinogenesis, we performed an in vitro natural killer (NK) cell cytotoxicity assay against 66cl4 tumors and found greater susceptibility to NK killing in Bptf knockdown tumors.