Metástases em ovário geralmente se apresentam como o primeiro sinal de doença, com o tumor primário não sendo imediatamente reconhecido. A diferenciação mucinosa é o fenótipo mais comum entre essas metástases. Em algumas situações, quando essa apresentação está associada a carcinomas metastáticos capazes de simular tumores ovarianos primários, essa configuração pode levar até mesmo patologistas experientes a diagnosticar incorretamente um depósito secundário como uma neoplasia primária. A maioria dos casos problemáticos pode ser resolvida correlacionando-se os dados clínicos, as características macroscópicas, os critérios de histologia e o perfil imuno-histoquímico, mas sempre haverá alguns casos com características sobrepostas. Levando-se em conta que a invasão linfovascular conspícua é uma das características que favorecem metástase, nós hipotetizamos que diferentes padrões de microdensidade vascular intratumoral poderiam nos ajudar na definição da origem primária ou secundária do tumor. Um total de 124 casos de tumores mucinosos de ovário foram selecionados, apresentando histologia \"borderline\" e maligna. Eles foram separados em dois grupos (primários ou metastáticos), classificados de acordo com as informações clínicas disponíveis, características macroscópicas e microscópicas, e perfil imuno-histoquímico realizado em amostras de tumores em microarranjo de tecido (TMA). A densidade vascular linfática (DVL) foi analisada quantificando-se espaços vasculares intratumorais identificados pela podoplanina, um marcador endotelial linfático. De acordo com os nossos resultados a DVL foi maior nas neoplasias primárias do que nas secundárias, mas, após análise multivariada, os melhores preditores de um tumor ser secundário foram tamanho de 10,0 cm ou menos (OR 9,4; IC 95% 1,2-69,2), bilateralidade (OR 51,5; IC 95% 7,1-370,2) e negatividade para CK7 (OR 64,8; IC 95% 9,4- 447). De acordo com o conhecimento atual, a disseminação metastática não é um evento aleatório, mas um processo de várias etapas complexas e sequenciais, altamente organizado e tecido específico. É importante aprofundar a relação entre as células tumorais e seu microambiente porque as características moleculares envolvidas podem ser uma possível fonte de novos marcadores que podem permitir uma categorização mais precisa dos tumores mucinosos nos ovários / Ovarian metastases commonly present as the first sign of the disease, with the primary tumor not being immediately recognized. Mucinous differentiation is the most common phenotype among those metastases. In particular situations, when compounded by the known metastatic carcinomas capable of simulate primary tumors, this setting can lead even experienced pathologists to diagnose incorrectly a secondary deposit as a primary neoplasm. Correlating clinical data, macroscopic features, histology criteria and immunohistochemistry profile, can solve the majority of those problematic cases, but there will always be some cases in a gray zone with superimposed characteristics. Since conspicuous lymphovascular invasion is one of the characteristics favoring metastases, we hypothesized if different patterns of intratumoral vascular microdensity can help us in defining the tumor origin. A total of 124 cases of mucinous tumors in ovary were selected, presenting borderline and malignant histology. They were separated in two groups (primary and metastatic) classified according to the available clinical data, gross and microscopic features, and immunohistochemistry profile performed in tumor samples in tissue microarrays (TMA). The lymphatic vascular density (LVD) was analyzed using podoplanin, a lymphatic endothelial marker. According to our results LVD was greater in primary than in secondary neoplasms, but after multivariate analysis, the best predictors of a secondary deposit tumor were size 10,0 cm or less (OR 9.4; CI 95% 1.2- 69.2), bilaterality (OR 51.5; CI 95% 7.1-370.2) and CK7 negativity (OR 64.8; CI 95% 9.4-447). According to actual knowledge metastatic dissemination is not a random event, but a complex and sequential multistep process, highly organized and tissue-specific. It is important to go deeper into the relation between tumor cells and their microenvironment because its molecular features may be a possible source of new markers that could allow a more precise categorization of mucinous tumors in the ovaries

No description available.

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the Philadelphia chromosome (Ph) that arises from a reciprocal translocation between chromosomes 9 and 22, thereby resulting in the formation of the chimeric BCR-ABL oncogene encoding a constitutively activated tyrosine kinase. BCR-ABL tyrosine kinase inhibitors (TKIs) induce a complete hematologic and cytogenetic response in the majority of chronic phrase CML patients. However, TKIs cannot efficiently eradicate leukemia stem cells (LSCs) because of the insensitivity of LSCs to TKIs. Therefore, developing new strategies to target LSCs is necessary and critical for curing CML, and success of this approach depends on further understanding the molecular mechanisms by which LSCs survive and are maintained. In Chapter I, I briefly introduce CML disease, BCR-ABL oncoprotein, and TKIs. I also describe the identification and features of LSCs. Several key pathways in LSCs including Wnt/ß-catenin, hedgehog, FoxO, Bcl6 and HIF1, are discussed. I also propose our strategy to identify unique molecular pathways that are important for LSCs but not their normal stem cell counterparts. In Chapter II, I describe our finding about the function of the positive regulator, HIF1α, in CML development and LSC survival. I show that loss of HIF1α impairs the maintenance of CML through impairing cell cycle progression and inducing apoptosis of LSCs, and I also report that p16Ink4a and p19Arf mediate the effect of HIF1α on LSCs, as knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α-/- LSCs. As detailed in Chapter III and IV, through comparing the global gene expression profiles of LSCs and HSCs, I find two novel regulators, Blk and Scd1, which act as tumor suppressors in CML development. In Chapter III, I show that Blk is markedly down-regulated by BCR-ABL in LSCs, and that c-Myc and Pax5 mediate this down-regulation. Deletion of Blk accelerates CML development; conversely, Blk overexpression significantly delays the development of CML and impairs the function of LSCs. I also demonstrate that p27, as a downstream effector, is involved in the function of Blk in LSCs. Blk also functions as a tumor suppressor in human CML stem cells, and inhibits the colony-forming ability of human CML cells. In Chapter IV, I investigate the function of another negative regulator, Scd1, in CML LSCs, and find that expression of Scd1 is down-regulated in mouse LSCs and human CML cells. We report that Scd1 acts as a tumor suppressor in CML, as loss of Scd1 causes acceleration of CML development and overexpression of Scd1 delays CML development. Using a colony-forming assay, I demonstrate that Scd1 impairs the maintenance of LSCs due to the change of expression of Pten, p53 and Bcl2. Importantly, I find that both Blk and Scd1 do not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Taken together, our findings demonstrate that HIF1α is required for the maintenance of CML LSCs, and conversely that Blk and Scd1 suppress the function of LSCs, suggesting that combining TKI treatment with specific targeting of LSCs will be necessary for curing CML.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes a life-long latent infection of B cells. It is usually asymptomatic in healthy individuals; however, individuals with compromised immunity often develop EBV-induced lymphoma. EBV also encodes potential oncogenes that can contribute to tumorigenesis. Therefore, vaccine and immunotherapeutic strategies targeting EBV are desirable. Recent studies have shown that infusion of EBV-specific CD8+T cells can elicit remission of lymphomas arising after administration of immunosuppressive drugs during transplantation, suggesting an important role for T cells in the prevention of EBV-induced malignancy. A better understanding of the cellular immune components involved in the control of EBV will aid in the development of methods to prevent infection and/or treat EBV-associated disease. While EBV infection is usually acquired asymptomatically during childhood, primary infection of adolescents and young adults can result in an illness termed acute infectious mononucleosis (AIM). Because of the characteristic symptoms of the illness, individuals with AIM can be readily identified and diagnosed with acute EBV infection. Thus, primary CD4+ and CD8+ T cell responses against the virus can be evaluated. It has been previously found that there is a marked expansion of lytic EBV protein-specific CD8+ T cells early during AIM, with delayed detection of lower frequencies of latent EBV protein-specific CD8+ T cells. The magnitude and specificity of CD4+T cell responses during AIM has been less well characterized. This thesis dissertation presents data from both functional assays and direct staining experiments documenting the timing, magnitude, and antigen-specificity of CD4+ T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4+ T cells were readily detected by intracellular IFN-γ production at presentation with AIM and declined rapidly thereafter. Blood EBV load was also quantitated and found to decrease over time following AIM. By contrast, CD8+T cell IFN-y responses remained high for several weeks following presentation with AIM. Direct staining of lytic epitope-specific CD4+ T cells during AIM revealed high frequencies of virus-specific cells with low proliferative and IFN-γ-producing potential. Blood EBV load in these patients was persistently high through 6 wk following AIM. These data suggest a relationship between high EBV load during acute infection and impaired EBV-specific CD4+ T cell responses, which are compatible with impaired CD4+ T cell responses reported during high viremia associated with other viral infections. This may represent a mechanism by which persistent viruses, such as EBV, are able to establish a life-long infection in their hosts.

DNA damage response pathways are a complicated network of proteins that function to remove and/or reverse DNA damage. Following genetic insult, a signal cascade is generated, which alerts the cell to the presence of damaged DNA. Once recognized, the damage is either removed or the damaged region is excised, and the original genetic sequence is restored. However, when these pathways are defective the cell is unable to effectively mediate the DNA damage response and the damage persists unrepaired. Thus, the proteins that maintain the DNA damage response pathway are critical in preserving genomic stability. One essential DNA repair protein is the Breast Cancer Associated gene, BRCA1. BRCA1 is essential for mediating the DNA damage response, facilitating DNA damage repair, and activating key cell cycle checkpoints. Moreover, mutations in BRCA1 lead to a higher incidence of breast and ovarian cancer, highlighting the importance of BRCA1 as a tumor suppressor. In an effort to better understand how BRCA1 carried out these functions, researchers sought to identify additional BRCA1 interacting proteins. This led to the identification of several proteins including the BRCA1 Associated C-terminal Helicase, BACH1. Due to the direct interaction of BACH1 with a region of BRCA1 essential for DNA repair and tumor suppression, it was speculated that BACH1 may help support these BRCA1 function(s). In fact, initial genetic screenings confirmed that mutations in BACH1 correlated not only with hereditary breast cancer, but also with defects in DNA damage repair processes. The initial correlation between BACH1 and cancer predisposition was further confirmed when mutations in BACH1 were identified in the cancer syndrome Fanconi anemia (FA) (complementation group FA-J), thus giving BACH1 its new name FANCJ. These findings supported a previously established link between the FA and BRCA pathways and between FA and DNA repair. In particular, we demonstrated that similar to other FA/BRCA proteins, suppression of FANCJ lead to a substantial decrease in homologous recombination and enhanced both the cellular sensitivity to DNA interstrand cross-linking agents and chromosomal instability. What remained unknown was specifically how FANCJ functioned and whether these functions were dependent on its interaction with BRCA1 or other associated partners. In fact, we identified that FANCJ interacted directly with the MMR protein MLH1. Moreover, we found that the FANCJ/BRCA1 interaction was not required to correct the cellular defects in FA-J cells, but rather that the FANCJ/MLH1 interaction was required. Although both the FA/BRCA and MMR pathways undoubtedly mediate the DNA damage response, there was no evidence to suggest that these pathways were linked, until recently. Our findings not only indicate a physical link between these pathways by protein-protein interaction, but also demonstrated a functional link.

Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.

The graduation thesis is focused on Lymphedema patientś problems. It addresses how the patient values his or her own health state. The differences between a patientś health state before therapy and after finishing its reduction phase have been compared. The following aspect have been valued: limited movement, pain and mental stress. All of them were improved after the reduction phase. Thus Hypothesis 1 has been confirmed: "At the end of the Reduction Phase of Lymphedema therapy the overal health state of the patient is improved." Out of above meditioned three aspect, the mental stress improved at a lowest rate. Differences between patients with and without good social background were demonstrated. Patients with bad social background valued mental stress caused by Lymphedema before and after the therapy as significantly higher then patients with good social background. Thus Hypothesis 2 was also confirmed: "Patients with good social background cope with their disease in a better way." In order to test the hypotheses the quantitative research was done. The technique was standardised integration.

Made available in DSpace on 2014-08-20T14:30:10Z (GMT). No. of bitstreams: 1 Dissertacao_alessandro_menna_alves.pdf: 813449 bytes, checksum: 43f43131b0d8e54633d172ea87ffa722 (MD5) Previous issue date: 2013-03-18 / This dissertation was divided into two distinct works, which can be summarized as follows: Article 1: The oral squamous cell carcinoma (OSCC) is the most prevalent malignance in mouth, being an important public health problem. The aim of this study was to evaluate the clinical and epidemiological profile of the OSCC cases registered in a center of clinical and histopathological diagnosis, located in Southern Brazil. Eight hundred and six individuals with OSCC and its variants were included in this study, over 1959-2012 period. The variables recorded from the files were: age, gender, skin color, tumor location, size and evolution time of the lesions (referred by the patients), as well as, the presence of pain lymph nodes, habits of tobacco and alcohol, and also the profession. OSSC was more frequent in males (76.6%), with the majority of cases distributed between 51 and 70 years old (53.9%). The most prevalent sites were lower lip vermilion [23.3% (20.4; 26.4)], followed by lateral border/ventral surface of the tongue [20.2% (17.5; 23.2)], gingiva/alveolar ridge [18.1% (15.5; 21.0)], and floor of the mouth [14.9% (12.5; 17.5)]. A strong association between outdoor occupation and OSCC in lower lip vermilion was found. The OSCC lesions located in tongue, gingiva/alveolar ridge and floor of the mouth were commonly more painful, bigger than 2 cm, and frequently presenting lymph nodes involvement. Most of the results confirm the data from literature. Mouth self-examination should be recommended and campaigns of prevention and early detection of OSCC should be periodically performed in order to increase people s feelings of personal risk. Article 2: Inside the tumor microenvironment (TM) the neoplastic cells are in dynamic crosstalk with the vascular and lymphatic endothelial cells in order to allow the tumor to growth and metastasize. Hypothesizing that there is a crosstalk between lymphatic endothelial cells and tumor cells from squamous cell carcinoma (SCC) that plays an important role in metastasis, we aimed to identify potential interactions between lymphatic endothelial cells and tumor cells lines from SCCs, through some in vitro assays. Primary adult human dermal lymphatic microvascular endothelial cells (HMVECs) and the human head and neck SCC cell lines like A431, UM-SCC-1, UM-SCC-22A and UM-SCC-22B were cultured in their specific media. UM-SCC cells lines were treated with rhIL-6, being VEGF-C expression checked by Elisa. Baseline IL-6 was evaluated in HMEVCs using the same assay. Also the IL-6 receptor (IL-6R) was analyzed by Western blot in UM-SCC cells. Conditioned media from HMVECs were prepared with different treatments and incubated with SCC A431 cells, in order to verify the MMPs enzymatic activities by gelatin zymography. Our results demonstrated that there are interactions between tumor cells and LECs, since the LECs-CM were able to enhance MMP-2 gelatinolytic activity. Moreover, we showed that LECs secrete IL-6, and different SCC lines have receptors for this cytokine. Therefore, our results indicate some potential interactions between LECs and TCs, being necessary other studies to elucidate the involved signaling pathways / Esta dissertação foi dividida em dois trabalhos distintos, os quais podem ser resumidos da seguinte maneira: Artigo 1: O carcinoma espinocelular oral (CEO) é o tumor maligno mais prevalente na cavidade oral, sendo um importante problema de saúde pública. O objetivo deste estudo foi avaliar o perfil clínico e epidemiológico dos casos registrados de CEO em um centro de diagnóstico clínico e histopatológico, localizado no Sul do Brasil. Oitocentos e seis indivíduos com CEO e suas variantes histológicas foram incluídos neste estudo, num período entre 1959 e 2012. As variáveis anotadas dos arquivos foram: idade, sexo, cor da pele, sítio, tamanho, tempo de evolução (relatado pelo paciente), assim como a presença de dor, linfonodos palpáveis, hábitos de tabagismo e etilismo, e a profissão. CEO foi mais prevalente em homens (76,6%), com a maioria dos casos distribuídos entre os 51 e 70 anos de idade (53,9%). Os sítios mais prevalentes foram vermelhão do lábio inferior [23,3% (20,4; 26,4)], seguido por borda lateral/ventre de língua [20,2% (17,5; 23,2)], gengiva/rebordo alveolar [18,1% (15,5; 21,0)], e assoalho bucal [14,9% (12,5; 17,5)]. Foi encontrada uma forte associação entre ocupações ao ar livre e CEO de vermelhão de lábio inferior. As lesões localizadas na língua, gengiva/rebordo alveolar e assoalho bucal foram comumente mais dolorosas, maiores que 2 cm, a frequentemente apresentavam envolvimento de linfonodos. A maioria dos nossos resultados confirmam os dados da literatura. O autoexame bucal deveria ser recomendado e campanhas de prevenção e detecção precoce do CEO deveriam ser realizadas periodicamente na tentativa de aumentar o sentimento pessoal em relação ao CEO. Artigo 2: Dentro do microambiente tumoral (MT), as células neoplásicas estão numa constante crosstalk com células endoteliais linfáticas (CELs) e sanguíneas a fim de permitir o crescimento tumoral e metástase. Supondo que haja um crosstalk entre as CELs e as células do carcinoma espinocelular (CE) que exerce um importante papel na metástase, nosso objetivo foi identificar potenciais interações entre as CELs e linhagens celulares de CE, através de alguns ensaios in vitro. Células endoteliais linfáticas primárias adultas humanas da microvasculatura dérmica (HMVECs) e as linhagens de CE A431, UM-SCC-1, UM-SCC-22A e UM-SCC-22B foram cultivadas nos seus meios específicos. UM-SCC foram tratadas com rhIL-6, sendo a expressão de VEGF-C verificada por Elisa. Produção natural de IL-6 pelas HMVECs foi avaliada da mesma maneira. A presença de receptor de IL-6 (IL-6R) foi analisada por Western Blot nas linhagens UM-SCC. Meios condicionados(MC) das HMVECs foram preparados com diferentes tratamentos e incubados com a linhagem A431, a fim de verificar a atividade genatinolítica das MMPs por zimografia. Nossos resultados demonstraram que há interações entre as células tumorais e as CELs, uma vez que MC-CELS foram capazes de aumentar a atividade genatinolítica da MMP-2. Além disso, nós mostramos que as CELs secretam IL-6, e diferentes linhagens de CE possuem receptores para esta citocina. Sendo assim, nossos resultados indicam potenciais interações entre as LECs e as células tumorais, sendo necessário outros estudos para elucidar as vias de sinalização envolvidas

The human Philadelphia chromosome (Ph) arises from a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. The resulting chimeric BCR-ABLoncogene encodes a constitutively activated, oncogenic tyrosine kinase that induces chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitor (TKI), imatinib mesylate, induces a complete hematologic and cytogenetic response in the majority of CML patients, but is unable to completely eradicate BCR-ABL–expressing leukemic cells, suggesting that leukemia stem cells are not eliminated. Over time, patients frequently become drug resistant and develop progressive disease despite continued treatment. Two major reasons cause the imatinib resistance. The first one is the BCR-ABL kinase domain mutations which inhibit the interaction of BCR-ABL kinase domain with imatinib; the second one is the residual leukemia stem cells (LSCs) in the patients who are administrated with imatinib. To overcome these two major obstacles in CML treatment, new strategies need further investigation. As detailed in Chapter II, we evaluated the therapeutic effect of Hsp90 inhibition by using a novel water-soluble Hsp90 inhibitor, IPI-504, in our BCR-ABL retroviral transplantation mouse model. We found that BCR-ABL mutants relied more on the HSP90 function than WT BCR-ABL in CML. More interestingly, inhibition of HSP90 in CML leukemia stem cells with IPI-504 significantly decreases the survival and proliferation of CML leukemia stem cells in vitro and in vivo. Consistent with these findings, IPI-504 treatment achieved significant prolonged survival of CML and B-ALL mice. IPI-504 represents a novel therapeutic approach whereby inhibition of Hsp90 in CML patients and Ph+ ALL may significantly advance efforts to develop a cure for these diseases. The rationale underlying the use of IPI-504 for kinase inhibitor–resistant CML has implications for other cancers that display oncogene addiction to kinases that are Hsp90 client proteins. Although we proved that inhibition of Hsp90 could restrain LSCs in vitro and in vivo, it is still unclear how to define specific targets in LSCs and eradicate LSCs. In Chapter III, we took advantage of our CML mouse model and compared the global gene expression signature between normal HSCs and LSCs to identify the downregulation of Pten in CML LSCs. CML develops faster when Pten is deleted in Ptenfl/fl mice. On the other hand, Pten overexpression significantly delays the CML development and impairs leukemia stem cell function. mTOR is a major downstream of Pten-Akt pathway and it is always activated or overepxressed when Pten is mutated or deleted in human cancers. In our study, we found that inhibition of mTOR by rapamycin inhibited proliferation and induced apoptosis of LSCs. Notably, our study also confirmed a recent clinical report that Pten has been downregulated in human CML patient LSCs. In summary, our results proved the tumor suppressor role of Pten in CML mouse model. Although the mechanisms of Pten in leukemia stem cells still need further study, Pten and its downstream, such as Akt and mTOR, should be more attractive in LSCs study.

Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive. First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells. Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model. Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients. Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies.