Claudia Huettner - Academia.edu (original) (raw)
Papers by Claudia Huettner
Frontiers in Medicine, Nov 9, 2018
PubMed, 1994
Interleukin 10 (Il-10) was initially discovered on the basis of its ability to suppress cytokine ... more Interleukin 10 (Il-10) was initially discovered on the basis of its ability to suppress cytokine synthesis. Additionally, it can exert immunosuppressive effects on a variety of cell types. Since patients with malignant gliomas present with a general impairment of the immune system, we sought to investigate if IL-10 is expressed in the glioma tissue. Using RT-PCR, IL-10 mRNA levels were determined in 37 glial tumors of different grades including 2 recurrencies, 3 specimens from normal brain tissue and 3 glioblastoma cell lines. Expression of IL-10 mRNA was demonstrable in all tumors as well as in normal brain. High grade tumors and recurrent cases expressed significantly higher amounts of IL-10 specific mRNA compared to low grade tumors, while 2 out of 3 cell lines showed only weak constitutive expression. We suggest, that IL-10 may contribute to the progression of astrocytomas by allowing the tumor cells to attenuate the T-cell immune response and evade immune detection.
Leukemia, May 14, 2013
The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the... more The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug-resistance. Altered expression of the anti-apoptotic Bcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally been demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacologic antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34 + progenitors but not those from healthy donors, regardless of drug-resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacologic approach for patients undergoing blastic transformation.
PubMed, Jan 15, 2002
The transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is important in the t... more The transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is important in the terminal differentiation of granulocytes, hepatocytes, and adipocytes, and recurrent mutations of C/EBPalpha were described in acute myeloid leukemia. In the lung, C/EBPalpha is expressed in bronchial cells and type II pneumocytes. Abnormal proliferation of the latter cell type was reported in C/EBPalpha knockout mice. We determined the expression of C/EBPalpha by Northern blot analysis in 30 lung cancer cell lines and found significant down-regulation in 24 cell lines. Immunohistochemical study of primary tumor specimens showed undetectable or low expression of C/EBPalpha in 23 of 53 specimens. Its expression was more frequently down-regulated in adenocarcinoma and poorly differentiated cancer specimens than in squamous cell cancers. A higher frequency of reduced expression was found in more advanced stages. To investigate the consequences of C/EBPalpha expression in lung cancer cells, we stably transfected two cell lines that do not express the gene (Calu1 and H358) with a plasmid allowing for induction of C/EBPalpha protein expression. Induction of C/EBPalpha led to significant growth reduction attributable to proliferation arrest, morphological changes characteristic of differentiation, and apoptosis. These results suggest that C/EBPalpha is down-regulated in a large proportion of lung cancers and that it has growth-inhibitory properties in airway epithelial cells. Genetic analysis of the C/EBPalpha gene is in progress to fully evaluate its role as a novel tumor suppressor in lung cancer.
PubMed, Feb 1, 1995
Interleukin 10 (IL-10) was initially discovered on the basis of its ability to suppress cytokine ... more Interleukin 10 (IL-10) was initially discovered on the basis of its ability to suppress cytokine synthesis. Additionally, it can exert immunosuppressive effects on a variety of cell types. Because patients with malignant gliomas present with a general impairment of the immune system, we investigated IL-10 expression in the glioma tissue. Because expression of IL-10 and IL-6 is associated in hematopoietic cells and IL-6 can act as an autocrine growth stimulator for glioblastoma cell lines, we looked in addition for a relationship between IL-10 and IL-6 expression. Using a quantitative reverse transcriptase polymerase chain reaction, IL-10 and IL-6 mRNA levels were determined in 37 glial tumors of different grades including 2 recurrencies, 3 specimens from normal brain tissue, and 3 glioblastoma cell lines. Expression of IL-10 mRNA was demonstrable in all tumors as well as in normal brain. High grade tumors and recurrent cases expressed significantly higher amounts of IL-10-specific mRNA compared with low grade tumors, whereas 2 of 3 cell lines showed only weak constitutive expression, mRNA for IL-6 was found in 86.5% of all gliomas with a correlation concerning the expression levels for both cytokines in 69% of gliomas. We suggest that IL-10 may contribute to the progression of astrocytomas by suppressing the patient's immune response, whereas IL-6 provides an additional growth advantage.
Blood, Nov 16, 2007
While treatment with tyrosine kinase inhibitors is highly successful for patients diagnosed in th... more While treatment with tyrosine kinase inhibitors is highly successful for patients diagnosed in the chronic phase of chronic myeloid leukemia, these drugs are inefficient for BCR/ABL associated B-cell acute lymphocytic leukemia (B-ALL). Therefore, it is necessary to identify molecular targets downstream of BCR/ABL to develop additional therapeutic approaches. Cells transformed by BCR/ABL are resistant to a wide variety of apoptotic stimuli and therapeutic strategies aimed at reinstating the apoptotic pathway appear as an attractive concept. Bcl-xL is an antiapoptotic member of the Bcl-2 family of proteins and studies employing cell lines, as well as primary cells have linked BCR/ABL expression with increased levels of Bcl-xL, resulting in resistance to chemotherapeutic agents. To define the role of Bcl-xL in BCR/ABL associated B-ALL, we generated two inducible transgenic mouse models. In the first model, BCR/ABL and loss of Bcl-x expression are co-induced, and in the second model, leukemia is induced with expression of Bcl-xL protein well above the levels found in wildtype lymphoblasts. Surprisingly, we found that deletion of Bcl-xL did not inhibit leukemogenesis or affect apoptosis. Bcl-x deficient B-ALL mice rapidly succumbed to a B-ALL like disease with Bcl-x deficient B-ALL animals being moribund as early as 17 days after induction. By day 28, all mice (n=10) had died or had to be euthanized. Necropsy of animals suffering from Bcl-x deficient leukemia revealed massive lymphadenopathy, pleural effusion, and splenomegaly. While loss of Bcl-x in our B-ALL model led to a more severe phenotype with considerable tumor burden, no statistically significant difference was found between the survival time in Bcl-x deficient and wild type B-ALL animals due to development of pleural effusion in both models. The most prominent difference was the presence of mitotic figures in the peripheral blood, lymph node, and spleen of Bcl-x deficient B-ALL animals, suggestive of increased proliferation of Bcl-x deficient lymphoblasts. Cell cycle analysis of leukemic cells isolated from pleural effusion and spleen of Bcl-x deficient B-ALL mice demonstrated a significant increase of cells in S/G2/M phase (p ≤ 0.05) compared to wildtype lymphoblasts. Thus, loss of Bcl-xL results in increased passage through the cell cycle, while expression of the protein limits the proliferation rate. To test this hypothesis, we generated a second model in which Bcl-xL is expressed at higher levels than in wild type lymphoblasts. Overexpression of Bcl-xL in BCR/ABL positive mice led to reduced proliferation as significantly fewer leukemic cells were present in the S phase than in controls substantiating a role for in Bcl-xL proliferation of lymphoblasts. Initial studies performed to determine the mechanisms by which loss of Bcl-x leads to increased proliferation suggest that the protein may indirectly regulate stability of p27Kip1. Our data show that cells from Bcl-x deficient B-ALL mice in G1 and S phase contain less p27, as a consequence of proteosomal degradation. Clearly, our model systems demonstrate an unexpected and novel role for Bcl-xL in the context of BCR/ABL associated B-ALL. Ongoing studies are aimed at the identification of the mechanism and molecules through which Bcl-xL is linked to cell cycle and proliferation of BCR/ABL transformed lymphoblasts.
Molecular and Cellular Biology, Jul 1, 1998
The transcription factor CCAAT/enhancer binding protein ␣ (C/EBP␣) regulates a number of myeloid ... more The transcription factor CCAAT/enhancer binding protein ␣ (C/EBP␣) regulates a number of myeloid cell-specific genes. To delineate the role of C/EBP␣ in human granulopoiesis, we studied its expression and function in human primary cells and bipotential (granulocytic/monocytic) myeloid cell lines. We show that the expression of C/EBP␣ initiates with the commitment of multipotential precursors to the myeloid lineage, is specifically upregulated during granulocytic differentiation, and is rapidly downregulated during the alternative monocytic pathway. Conditional expression of C/EBP␣ alone in stably transfected bipotential cells triggers neutrophilic differentiation, concomitant with upregulation of the granulocyte-specific granulocyte colonystimulating factor receptor and secondary granule protein genes. Moreover, induced expression of C/EBP␣ in bipotential precursors blocks their monocytic differentiation program. These results indicate that C/EBP␣ serves as a myeloid differentiation switch acting on bipotential precursors and directing them to mature to granulocytes.
Journal of Clinical Oncology, May 20, 2015
e17508 Background: Evaluation of patient tumors by large NGS gene panels is becoming routine at l... more e17508 Background: Evaluation of patient tumors by large NGS gene panels is becoming routine at large academic cancer centers. However, adoption and utilization of these diagnostic tools has not been studied in the community oncology practice setting. Matching a patient’s tumor mutational profile with targeted agents is the primary goal of personalized medicine in all treatment settings. The number of therapies entering clinical trials is continually increasing and these trials are becoming widely available in community oncology practices. Unlike academic centers where institutional funds typically cover NGS testing, community setting testing is reimbursed by commercial and government payers. We evaluated clinical utilization, performance, and payer characteristics in 100 sequential large NGS panels ordered by community practice oncologists. Methods: 100 sequential NGS gene panel results, obtained from community practice oncology groups, were evaluated for tumor type, stage and specimen source. Tumor prof...
Journal of Clinical Oncology, May 20, 2015
Blood, Nov 16, 2005
The size of the Natural Killer (NK) cell pool is maintained through production and subsequently e... more The size of the Natural Killer (NK) cell pool is maintained through production and subsequently export from the bone marrow, peripheral survival and proliferation, and ultimately cell death. While there exists considerable knowledge about developmental stages of lymphoid, myeloid and erythroid cells, comparably little is known about NK cell intermediates and the genes required for their development. Most of the models of NK cell differentiation have been based on in vitro culture systems where NK cells could be generated from multipotent HSC precursors. However, this approach suffers from the problems inherent to in vitro cell manipulations. We have utilized conditional gene targeting in adult mice to examine the role of the bcl-x gene in the development of NK cells in bone marrow and after their export to the spleen. Bcl-x is an important member of the anti-apoptotic member of the Bcl-2 gene family, and a critical role for this gene in the survival of hematopoietic cells was demonstrated in bcl-x-deficient mice causing embryonic death due to massive apoptosis of immature hematopoietic cells and of neurons. Conditional deletion in erythroid cells lead to hemolytic anemia and extensive splenomegaly. Furthermore, bcl-x is critical for the maturation of pre-B cells to the pro B cell stage, while it is not essential for the development of effector and memory T cells. We have conditionally deleted the gene in the stem cells of adult mice by cross breeding them with the Mxi-cre deleter strain, which allows for induced expression of cre recombinase by injection with pIpC. As early as 9 days after the first injection of pIpC, the number of NK cells in the bone marrow of mice started to decline as demonstrated by multi-color FACS analysis staining for IL2 Receptor beta (CD122) and NKG2D, among other markers. Cultures of bone marrow and spleen cells in the presence of cytokines to generate lymphokine activated killer cells failed, while no such effect was observed in cultures from Mxi-cre mice that were subjected to pIpC injections and carried along as controls. Analysis of animals after 3 weeks of pIpC administration revealed absence of NK cell precursors in the bone marrow as demonstrated by the lack of CD122+/Lin- negative cells. This phenomenon was accompanied by a reduction in the number of mature NK cells in the spleen. To date, six stages of NK cell maturation are described with the acquisition of IL2 receptor beta expression marking commitment to the NK-cell lineage. IL2 Receptor beta as well as NKG2D are expressed throughout NK cell development and at all stages. In order to characterize the specific stage at which expression of bcl-x is essential for NK cell maturation, we employed multi-color FACS analysis staining for CD122, NKG2D, CD49b, and the integrins CD43 and Mac-1 after depletion of lineage positive cells, followed by sorting for defined populations. Real Time PCR on sorted cells demonstrated that Bcl-x mRNA is highly expressed throughout all stages of NK cell development. Taken together, the gradual reduction in the number of NK cell precursors eventually leading to complete loss of this lineage in the bone marrow and peripheral sites suggests that bcl-x is indispensable for the development of NK cells presumably from the earliest time point of commitment to this lineage.
JAMIA Open
Objectives Describe an augmented intelligence approach to facilitate the update of evidence for a... more Objectives Describe an augmented intelligence approach to facilitate the update of evidence for associations in knowledge graphs. Methods New publications are filtered through multiple machine learning study classifiers, and filtered publications are combined with articles already included as evidence in the knowledge graph. The corpus is then subjected to named entity recognition, semantic dictionary mapping, term vector space modeling, pairwise similarity, and focal entity match to identify highly related publications. Subject matter experts review recommended articles to assess inclusion in the knowledge graph; discrepancies are resolved by consensus. Results Study classifiers achieved F-scores from 0.88 to 0.94, and similarity thresholds for each study type were determined by experimentation. Our approach reduces human literature review load by 99%, and over the past 12 months, 41% of recommendations were accepted to update the knowledge graph. Conclusion Integrated search and r...
Cancer Research, 2011
Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadel... more Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadelphia-positive (Ph+) CML progenitors to BCR-ABL1 kinase activity. However, CML quiescent hematopoietic stem cells (HSC) are TKI-resistant and represent an active disease reservoir. We hypothesize that this innate drug-resistance depends on inhibition of the tumor suppressor protein phosphatase 2A (PP2A). PP2A can be reactivated by FTY720, a drug that targets CML but not normal progenitors. Here we investigated the mechanism controlling survival/self-renewal of quiescent leukemic HSCs and their sensitivity to PP2A-activating drugs. Methods: HSCs from CML (n=68) and healthy (n=12) donors were FACS-isolated, and the biologic importance of PP2A inhibition and pharmacologic PP2A activation on their survival/self-renewal was assessed by BM serial transplantation; CFSE and Annexin-V staining; LTC-IC and CFC/replating assays; lentiviral shRNA/cDNA-transduction; LEF/TCF and proximity-ligation assa...
We showed previously that CCAAT/enhancer binding protein (C/ EBP ), a tissue-specific transcripti... more We showed previously that CCAAT/enhancer binding protein (C/ EBP ), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBP target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3 (HNF3 ), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBP . We found downregulation of HNF3 expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3 , as well as hypermethylation of the HNF3 promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3 expression. Conditional expression of HNF3 led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3 is a novel tumor suppressor in lung cancer. This is ...
Blood
CML is a clonal disorder of the pluripotent hematopoietic stem cell characterized by the sustaine... more CML is a clonal disorder of the pluripotent hematopoietic stem cell characterized by the sustained kinase activity of the BCR/ABL oncoprotein. We reported that the BCR/ABL-dependent and SET-mediated inhibition of protein phosphatase PP2A tumor suppressor activity is essential for the leukemogenic potential of CD34+ CML bone marrow progenitors, as molecular and pharmacologic restoration of PP2A inhibits the activity of BCR/ABL and that of several important regulators of cell survival/proliferation, thus resulting in marked apoptosis, impaired clonogenic potential and in vivo leukemogenesis of imatinib/dasatinib-sensitive and -resistant Ph(+), but not normal, CD34+ blasts and/or BCR/ABL+ mouse marrow progenitors. Here we show that SET-dependent suppression of PP2A activity is a common feature of Ph(+) progenitors (CMP and GMP) and imatinib/ dasatinib-insensitive CD34+/CD38- BCR/ABL+ (n=3) stem cells but not of the equivalent cell fractions from healthy individuals (n=3). To determine ...
Blood
Germline deletion of bcl-x demonstrated the importance of this gene for hematopoiesis. Bcl-x defi... more Germline deletion of bcl-x demonstrated the importance of this gene for hematopoiesis. Bcl-x deficient embryos failed to thrive due to massive apoptosis of immature hematopoietic and neural cells. Deletion of this gene in the adult has uncovered its requirement for the final maturation of erythroid cells, the transition of pre-B cells to the pro-B cell stage and recently, we showed an important role in the development of natural killer cells. While the role of bcl-x in hematopoietic stem cells (HSC) has yet to be addressed, a role for other members of anti-apoptotic bcl-2 family of proteins has been established. Overexpression of bcl-2 led to accumulation of HSC, whereas genetic deletion of mcl-1 resulted in ablation of HSC. In the present study, we determined by quantitative Real-Time PCR, that in addition to mcl-1, bcl-x is also highly expressed in hematopoietic stem cells, whereas the level of bcl-2 is threefold lower. Moreover, we found the expression of bcl-x to be down regulat...
Journal of Clinical Oncology
e24274Background: Immune checkpoint inhibitors (CPIs) induce prolonged tumor responses in a minor... more e24274Background: Immune checkpoint inhibitors (CPIs) induce prolonged tumor responses in a minority of patients with solid tumors including those with MSI-high, melanoma and NSCLC. Established pre...
Blood
962 Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem c... more 962 Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem cell (HSC) lodgment and maintenance. However microenvironmental interactions of leukemia stem cells (LSC) are poorly understood. Although chronic myelogenous leukemia (CML) results from HSC transformation by the BCR-ABL gene, the role of the microenvironment in modulating leukemia development is not known. We employed the SCL-tTA-BCR/ABL mouse model of CML to investigate the LSC interactions with the BM microenvironment. In this model, targeted expression of the BCR-ABL gene in murine HSC via a tet-regulated SCL promoter results in development of a chronic phase CML-like disorder. We have reported that LSC capacity is restricted to BCR-ABL+ cells with long-term hematopoietic stem cell (LTHSC) phenotype(LSK Flt3-CD150+CD48-) (Blood 2010 116:1212A). LSC numbers are reduced in the BM but increased in the spleen of CML mice compared with LTHSC from control mice, suggesting that LSC have alter...
Blood
Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hem... more Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hematopoietic stem cells (HSC) by the BCR-ABL oncogene. Using the tet-off system, we have generated double-transgenic mice in which BCR-ABL is expressed under the control of the murine SCL 3′ enhancer, which targets expression to the vast majority of HSC and progenitors. After induction of BCR-ABL, all mice developed progressive chronic neutrophilia and leukocytosis (20–40 K/ul), and the animals died or were sacrificed in moribund condition within 58+/−28 days. Upon necropsy, bone marrow granulocytic hyperplasia, splenomegaly as well as organ infiltration by leukemic cells (liver, kidney, lung, small intestine, skin) were found. In addition, 31% of the mice subsequently developed ALL or lymphomas. BCR-ABL mRNA and protein expression were demonstrated in the affected organs. Expression of the transactivating transgene tTA was high in HSC, CMP, and CLP, but low in GMP and MEP, as assessed by ...
Blood
Abl kinase inhibitors are the preferred treatment for Chronic Myelogenous Leukemia (CML) and they... more Abl kinase inhibitors are the preferred treatment for Chronic Myelogenous Leukemia (CML) and they are highly efficient at inducing remission but continued treatment is required as not all BCR/ABL positive cells are eradicated. Survival of tumor cells may be mediated by deregulation of apoptosis pathways as cells transformed by BCR/ABL are highly resistant to a variety of apoptotic stimuli. Resistance to drug-induced apoptosis in CD34+ CML patient cells and BCR/ABL+ cell lines is mediated by downstream signaling pathways activated by BCR/ABL. The signal transducer and activator of transcription 5 pathway which regulates the expression of the anti-apoptotic Bcl-x gene is constitutively activated in BCR/ABL-positive cells. Several lines of evidence led to the hypothesis that Bcl-x is a critical mediator in evasion of apoptosis required to sustain leukemia. In order to examine the in vivo role of Bcl-x in CML we generated a tetracycline-inducible transgenic mouse model in which BCR/ABL ...
Frontiers in Medicine, Nov 9, 2018
PubMed, 1994
Interleukin 10 (Il-10) was initially discovered on the basis of its ability to suppress cytokine ... more Interleukin 10 (Il-10) was initially discovered on the basis of its ability to suppress cytokine synthesis. Additionally, it can exert immunosuppressive effects on a variety of cell types. Since patients with malignant gliomas present with a general impairment of the immune system, we sought to investigate if IL-10 is expressed in the glioma tissue. Using RT-PCR, IL-10 mRNA levels were determined in 37 glial tumors of different grades including 2 recurrencies, 3 specimens from normal brain tissue and 3 glioblastoma cell lines. Expression of IL-10 mRNA was demonstrable in all tumors as well as in normal brain. High grade tumors and recurrent cases expressed significantly higher amounts of IL-10 specific mRNA compared to low grade tumors, while 2 out of 3 cell lines showed only weak constitutive expression. We suggest, that IL-10 may contribute to the progression of astrocytomas by allowing the tumor cells to attenuate the T-cell immune response and evade immune detection.
Leukemia, May 14, 2013
The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the... more The dismal outcome of blast crisis chronic myelogenous leukemia (CML-BC) patients underscores the need for a better understanding of the mechanisms responsible for the development of drug-resistance. Altered expression of the anti-apoptotic Bcl-xL has been correlated with BCR-ABL leukemogenesis; however, its involvement in the pathogenesis and evolution of CML has not been formally been demonstrated yet. Thus, we generated an inducible mouse model in which simultaneous expression of p210-BCR-ABL1 and deletion of bcl-x occurs within hematopoietic stem and progenitor cells. Absence of Bcl-xL did not affect development of the chronic phase-like myeloproliferative disease, but none of the deficient mice progressed to an advanced phenotype, suggesting the importance of Bcl-xL in survival of progressing early progenitor cells. Indeed, pharmacologic antagonism of Bcl-xL, with ABT-263, combined with PP242-induced activation of BAD markedly augmented apoptosis of CML-BC cell lines and primary CD34 + progenitors but not those from healthy donors, regardless of drug-resistance induced by bone marrow stromal cell-generated signals. Moreover, studies in which BAD or Bcl-xL expression was molecularly altered strongly support their involvement in ABT-263/PP242induced apoptosis of CML-BC progenitors. Thus, suppression of the antiapoptotic potential of Bcl-xL together with BAD activation represents an effective pharmacologic approach for patients undergoing blastic transformation.
PubMed, Jan 15, 2002
The transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is important in the t... more The transcription factor, CCAAT/enhancer binding protein alpha (C/EBPalpha) is important in the terminal differentiation of granulocytes, hepatocytes, and adipocytes, and recurrent mutations of C/EBPalpha were described in acute myeloid leukemia. In the lung, C/EBPalpha is expressed in bronchial cells and type II pneumocytes. Abnormal proliferation of the latter cell type was reported in C/EBPalpha knockout mice. We determined the expression of C/EBPalpha by Northern blot analysis in 30 lung cancer cell lines and found significant down-regulation in 24 cell lines. Immunohistochemical study of primary tumor specimens showed undetectable or low expression of C/EBPalpha in 23 of 53 specimens. Its expression was more frequently down-regulated in adenocarcinoma and poorly differentiated cancer specimens than in squamous cell cancers. A higher frequency of reduced expression was found in more advanced stages. To investigate the consequences of C/EBPalpha expression in lung cancer cells, we stably transfected two cell lines that do not express the gene (Calu1 and H358) with a plasmid allowing for induction of C/EBPalpha protein expression. Induction of C/EBPalpha led to significant growth reduction attributable to proliferation arrest, morphological changes characteristic of differentiation, and apoptosis. These results suggest that C/EBPalpha is down-regulated in a large proportion of lung cancers and that it has growth-inhibitory properties in airway epithelial cells. Genetic analysis of the C/EBPalpha gene is in progress to fully evaluate its role as a novel tumor suppressor in lung cancer.
PubMed, Feb 1, 1995
Interleukin 10 (IL-10) was initially discovered on the basis of its ability to suppress cytokine ... more Interleukin 10 (IL-10) was initially discovered on the basis of its ability to suppress cytokine synthesis. Additionally, it can exert immunosuppressive effects on a variety of cell types. Because patients with malignant gliomas present with a general impairment of the immune system, we investigated IL-10 expression in the glioma tissue. Because expression of IL-10 and IL-6 is associated in hematopoietic cells and IL-6 can act as an autocrine growth stimulator for glioblastoma cell lines, we looked in addition for a relationship between IL-10 and IL-6 expression. Using a quantitative reverse transcriptase polymerase chain reaction, IL-10 and IL-6 mRNA levels were determined in 37 glial tumors of different grades including 2 recurrencies, 3 specimens from normal brain tissue, and 3 glioblastoma cell lines. Expression of IL-10 mRNA was demonstrable in all tumors as well as in normal brain. High grade tumors and recurrent cases expressed significantly higher amounts of IL-10-specific mRNA compared with low grade tumors, whereas 2 of 3 cell lines showed only weak constitutive expression, mRNA for IL-6 was found in 86.5% of all gliomas with a correlation concerning the expression levels for both cytokines in 69% of gliomas. We suggest that IL-10 may contribute to the progression of astrocytomas by suppressing the patient's immune response, whereas IL-6 provides an additional growth advantage.
Blood, Nov 16, 2007
While treatment with tyrosine kinase inhibitors is highly successful for patients diagnosed in th... more While treatment with tyrosine kinase inhibitors is highly successful for patients diagnosed in the chronic phase of chronic myeloid leukemia, these drugs are inefficient for BCR/ABL associated B-cell acute lymphocytic leukemia (B-ALL). Therefore, it is necessary to identify molecular targets downstream of BCR/ABL to develop additional therapeutic approaches. Cells transformed by BCR/ABL are resistant to a wide variety of apoptotic stimuli and therapeutic strategies aimed at reinstating the apoptotic pathway appear as an attractive concept. Bcl-xL is an antiapoptotic member of the Bcl-2 family of proteins and studies employing cell lines, as well as primary cells have linked BCR/ABL expression with increased levels of Bcl-xL, resulting in resistance to chemotherapeutic agents. To define the role of Bcl-xL in BCR/ABL associated B-ALL, we generated two inducible transgenic mouse models. In the first model, BCR/ABL and loss of Bcl-x expression are co-induced, and in the second model, leukemia is induced with expression of Bcl-xL protein well above the levels found in wildtype lymphoblasts. Surprisingly, we found that deletion of Bcl-xL did not inhibit leukemogenesis or affect apoptosis. Bcl-x deficient B-ALL mice rapidly succumbed to a B-ALL like disease with Bcl-x deficient B-ALL animals being moribund as early as 17 days after induction. By day 28, all mice (n=10) had died or had to be euthanized. Necropsy of animals suffering from Bcl-x deficient leukemia revealed massive lymphadenopathy, pleural effusion, and splenomegaly. While loss of Bcl-x in our B-ALL model led to a more severe phenotype with considerable tumor burden, no statistically significant difference was found between the survival time in Bcl-x deficient and wild type B-ALL animals due to development of pleural effusion in both models. The most prominent difference was the presence of mitotic figures in the peripheral blood, lymph node, and spleen of Bcl-x deficient B-ALL animals, suggestive of increased proliferation of Bcl-x deficient lymphoblasts. Cell cycle analysis of leukemic cells isolated from pleural effusion and spleen of Bcl-x deficient B-ALL mice demonstrated a significant increase of cells in S/G2/M phase (p ≤ 0.05) compared to wildtype lymphoblasts. Thus, loss of Bcl-xL results in increased passage through the cell cycle, while expression of the protein limits the proliferation rate. To test this hypothesis, we generated a second model in which Bcl-xL is expressed at higher levels than in wild type lymphoblasts. Overexpression of Bcl-xL in BCR/ABL positive mice led to reduced proliferation as significantly fewer leukemic cells were present in the S phase than in controls substantiating a role for in Bcl-xL proliferation of lymphoblasts. Initial studies performed to determine the mechanisms by which loss of Bcl-x leads to increased proliferation suggest that the protein may indirectly regulate stability of p27Kip1. Our data show that cells from Bcl-x deficient B-ALL mice in G1 and S phase contain less p27, as a consequence of proteosomal degradation. Clearly, our model systems demonstrate an unexpected and novel role for Bcl-xL in the context of BCR/ABL associated B-ALL. Ongoing studies are aimed at the identification of the mechanism and molecules through which Bcl-xL is linked to cell cycle and proliferation of BCR/ABL transformed lymphoblasts.
Molecular and Cellular Biology, Jul 1, 1998
The transcription factor CCAAT/enhancer binding protein ␣ (C/EBP␣) regulates a number of myeloid ... more The transcription factor CCAAT/enhancer binding protein ␣ (C/EBP␣) regulates a number of myeloid cell-specific genes. To delineate the role of C/EBP␣ in human granulopoiesis, we studied its expression and function in human primary cells and bipotential (granulocytic/monocytic) myeloid cell lines. We show that the expression of C/EBP␣ initiates with the commitment of multipotential precursors to the myeloid lineage, is specifically upregulated during granulocytic differentiation, and is rapidly downregulated during the alternative monocytic pathway. Conditional expression of C/EBP␣ alone in stably transfected bipotential cells triggers neutrophilic differentiation, concomitant with upregulation of the granulocyte-specific granulocyte colonystimulating factor receptor and secondary granule protein genes. Moreover, induced expression of C/EBP␣ in bipotential precursors blocks their monocytic differentiation program. These results indicate that C/EBP␣ serves as a myeloid differentiation switch acting on bipotential precursors and directing them to mature to granulocytes.
Journal of Clinical Oncology, May 20, 2015
e17508 Background: Evaluation of patient tumors by large NGS gene panels is becoming routine at l... more e17508 Background: Evaluation of patient tumors by large NGS gene panels is becoming routine at large academic cancer centers. However, adoption and utilization of these diagnostic tools has not been studied in the community oncology practice setting. Matching a patient’s tumor mutational profile with targeted agents is the primary goal of personalized medicine in all treatment settings. The number of therapies entering clinical trials is continually increasing and these trials are becoming widely available in community oncology practices. Unlike academic centers where institutional funds typically cover NGS testing, community setting testing is reimbursed by commercial and government payers. We evaluated clinical utilization, performance, and payer characteristics in 100 sequential large NGS panels ordered by community practice oncologists. Methods: 100 sequential NGS gene panel results, obtained from community practice oncology groups, were evaluated for tumor type, stage and specimen source. Tumor prof...
Journal of Clinical Oncology, May 20, 2015
Blood, Nov 16, 2005
The size of the Natural Killer (NK) cell pool is maintained through production and subsequently e... more The size of the Natural Killer (NK) cell pool is maintained through production and subsequently export from the bone marrow, peripheral survival and proliferation, and ultimately cell death. While there exists considerable knowledge about developmental stages of lymphoid, myeloid and erythroid cells, comparably little is known about NK cell intermediates and the genes required for their development. Most of the models of NK cell differentiation have been based on in vitro culture systems where NK cells could be generated from multipotent HSC precursors. However, this approach suffers from the problems inherent to in vitro cell manipulations. We have utilized conditional gene targeting in adult mice to examine the role of the bcl-x gene in the development of NK cells in bone marrow and after their export to the spleen. Bcl-x is an important member of the anti-apoptotic member of the Bcl-2 gene family, and a critical role for this gene in the survival of hematopoietic cells was demonstrated in bcl-x-deficient mice causing embryonic death due to massive apoptosis of immature hematopoietic cells and of neurons. Conditional deletion in erythroid cells lead to hemolytic anemia and extensive splenomegaly. Furthermore, bcl-x is critical for the maturation of pre-B cells to the pro B cell stage, while it is not essential for the development of effector and memory T cells. We have conditionally deleted the gene in the stem cells of adult mice by cross breeding them with the Mxi-cre deleter strain, which allows for induced expression of cre recombinase by injection with pIpC. As early as 9 days after the first injection of pIpC, the number of NK cells in the bone marrow of mice started to decline as demonstrated by multi-color FACS analysis staining for IL2 Receptor beta (CD122) and NKG2D, among other markers. Cultures of bone marrow and spleen cells in the presence of cytokines to generate lymphokine activated killer cells failed, while no such effect was observed in cultures from Mxi-cre mice that were subjected to pIpC injections and carried along as controls. Analysis of animals after 3 weeks of pIpC administration revealed absence of NK cell precursors in the bone marrow as demonstrated by the lack of CD122+/Lin- negative cells. This phenomenon was accompanied by a reduction in the number of mature NK cells in the spleen. To date, six stages of NK cell maturation are described with the acquisition of IL2 receptor beta expression marking commitment to the NK-cell lineage. IL2 Receptor beta as well as NKG2D are expressed throughout NK cell development and at all stages. In order to characterize the specific stage at which expression of bcl-x is essential for NK cell maturation, we employed multi-color FACS analysis staining for CD122, NKG2D, CD49b, and the integrins CD43 and Mac-1 after depletion of lineage positive cells, followed by sorting for defined populations. Real Time PCR on sorted cells demonstrated that Bcl-x mRNA is highly expressed throughout all stages of NK cell development. Taken together, the gradual reduction in the number of NK cell precursors eventually leading to complete loss of this lineage in the bone marrow and peripheral sites suggests that bcl-x is indispensable for the development of NK cells presumably from the earliest time point of commitment to this lineage.
JAMIA Open
Objectives Describe an augmented intelligence approach to facilitate the update of evidence for a... more Objectives Describe an augmented intelligence approach to facilitate the update of evidence for associations in knowledge graphs. Methods New publications are filtered through multiple machine learning study classifiers, and filtered publications are combined with articles already included as evidence in the knowledge graph. The corpus is then subjected to named entity recognition, semantic dictionary mapping, term vector space modeling, pairwise similarity, and focal entity match to identify highly related publications. Subject matter experts review recommended articles to assess inclusion in the knowledge graph; discrepancies are resolved by consensus. Results Study classifiers achieved F-scores from 0.88 to 0.94, and similarity thresholds for each study type were determined by experimentation. Our approach reduces human literature review load by 99%, and over the past 12 months, 41% of recommendations were accepted to update the knowledge graph. Conclusion Integrated search and r...
Cancer Research, 2011
Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadel... more Background: The success of tyrosine kinase inhibitors (TKIs) depends on the addiction of Philadelphia-positive (Ph+) CML progenitors to BCR-ABL1 kinase activity. However, CML quiescent hematopoietic stem cells (HSC) are TKI-resistant and represent an active disease reservoir. We hypothesize that this innate drug-resistance depends on inhibition of the tumor suppressor protein phosphatase 2A (PP2A). PP2A can be reactivated by FTY720, a drug that targets CML but not normal progenitors. Here we investigated the mechanism controlling survival/self-renewal of quiescent leukemic HSCs and their sensitivity to PP2A-activating drugs. Methods: HSCs from CML (n=68) and healthy (n=12) donors were FACS-isolated, and the biologic importance of PP2A inhibition and pharmacologic PP2A activation on their survival/self-renewal was assessed by BM serial transplantation; CFSE and Annexin-V staining; LTC-IC and CFC/replating assays; lentiviral shRNA/cDNA-transduction; LEF/TCF and proximity-ligation assa...
We showed previously that CCAAT/enhancer binding protein (C/ EBP ), a tissue-specific transcripti... more We showed previously that CCAAT/enhancer binding protein (C/ EBP ), a tissue-specific transcription factor, is a candidate tumor suppressor in lung cancer. In the present study, we have performed a transcriptional profiling study of C/EBP target genes using an inducible cell line system. This study led to the identification of hepatocyte nuclear factor 3 (HNF3 ), a transcription factor known to play a role in airway differentiation, as a downstream target of C/EBP . We found downregulation of HNF3 expression in a large proportion of lung cancer cell lines examined and identified two novel mutants of HNF3 , as well as hypermethylation of the HNF3 promoter. We also developed a tetracycline-inducible cell line model to study the cellular consequences of HNF3 expression. Conditional expression of HNF3 led to significant growth reduction, proliferation arrest, apoptosis, and loss of clonogenic ability, suggesting additionally that HNF3 is a novel tumor suppressor in lung cancer. This is ...
Blood
CML is a clonal disorder of the pluripotent hematopoietic stem cell characterized by the sustaine... more CML is a clonal disorder of the pluripotent hematopoietic stem cell characterized by the sustained kinase activity of the BCR/ABL oncoprotein. We reported that the BCR/ABL-dependent and SET-mediated inhibition of protein phosphatase PP2A tumor suppressor activity is essential for the leukemogenic potential of CD34+ CML bone marrow progenitors, as molecular and pharmacologic restoration of PP2A inhibits the activity of BCR/ABL and that of several important regulators of cell survival/proliferation, thus resulting in marked apoptosis, impaired clonogenic potential and in vivo leukemogenesis of imatinib/dasatinib-sensitive and -resistant Ph(+), but not normal, CD34+ blasts and/or BCR/ABL+ mouse marrow progenitors. Here we show that SET-dependent suppression of PP2A activity is a common feature of Ph(+) progenitors (CMP and GMP) and imatinib/ dasatinib-insensitive CD34+/CD38- BCR/ABL+ (n=3) stem cells but not of the equivalent cell fractions from healthy individuals (n=3). To determine ...
Blood
Germline deletion of bcl-x demonstrated the importance of this gene for hematopoiesis. Bcl-x defi... more Germline deletion of bcl-x demonstrated the importance of this gene for hematopoiesis. Bcl-x deficient embryos failed to thrive due to massive apoptosis of immature hematopoietic and neural cells. Deletion of this gene in the adult has uncovered its requirement for the final maturation of erythroid cells, the transition of pre-B cells to the pro-B cell stage and recently, we showed an important role in the development of natural killer cells. While the role of bcl-x in hematopoietic stem cells (HSC) has yet to be addressed, a role for other members of anti-apoptotic bcl-2 family of proteins has been established. Overexpression of bcl-2 led to accumulation of HSC, whereas genetic deletion of mcl-1 resulted in ablation of HSC. In the present study, we determined by quantitative Real-Time PCR, that in addition to mcl-1, bcl-x is also highly expressed in hematopoietic stem cells, whereas the level of bcl-2 is threefold lower. Moreover, we found the expression of bcl-x to be down regulat...
Journal of Clinical Oncology
e24274Background: Immune checkpoint inhibitors (CPIs) induce prolonged tumor responses in a minor... more e24274Background: Immune checkpoint inhibitors (CPIs) induce prolonged tumor responses in a minority of patients with solid tumors including those with MSI-high, melanoma and NSCLC. Established pre...
Blood
962 Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem c... more 962 Specialized bone marrow (BM) microenvironmental niches are essential for hematopoietic stem cell (HSC) lodgment and maintenance. However microenvironmental interactions of leukemia stem cells (LSC) are poorly understood. Although chronic myelogenous leukemia (CML) results from HSC transformation by the BCR-ABL gene, the role of the microenvironment in modulating leukemia development is not known. We employed the SCL-tTA-BCR/ABL mouse model of CML to investigate the LSC interactions with the BM microenvironment. In this model, targeted expression of the BCR-ABL gene in murine HSC via a tet-regulated SCL promoter results in development of a chronic phase CML-like disorder. We have reported that LSC capacity is restricted to BCR-ABL+ cells with long-term hematopoietic stem cell (LTHSC) phenotype(LSK Flt3-CD150+CD48-) (Blood 2010 116:1212A). LSC numbers are reduced in the BM but increased in the spleen of CML mice compared with LTHSC from control mice, suggesting that LSC have alter...
Blood
Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hem... more Chronic myeloid leukemia (CML) is a malignant disorder originating from the transformation of hematopoietic stem cells (HSC) by the BCR-ABL oncogene. Using the tet-off system, we have generated double-transgenic mice in which BCR-ABL is expressed under the control of the murine SCL 3′ enhancer, which targets expression to the vast majority of HSC and progenitors. After induction of BCR-ABL, all mice developed progressive chronic neutrophilia and leukocytosis (20–40 K/ul), and the animals died or were sacrificed in moribund condition within 58+/−28 days. Upon necropsy, bone marrow granulocytic hyperplasia, splenomegaly as well as organ infiltration by leukemic cells (liver, kidney, lung, small intestine, skin) were found. In addition, 31% of the mice subsequently developed ALL or lymphomas. BCR-ABL mRNA and protein expression were demonstrated in the affected organs. Expression of the transactivating transgene tTA was high in HSC, CMP, and CLP, but low in GMP and MEP, as assessed by ...
Blood
Abl kinase inhibitors are the preferred treatment for Chronic Myelogenous Leukemia (CML) and they... more Abl kinase inhibitors are the preferred treatment for Chronic Myelogenous Leukemia (CML) and they are highly efficient at inducing remission but continued treatment is required as not all BCR/ABL positive cells are eradicated. Survival of tumor cells may be mediated by deregulation of apoptosis pathways as cells transformed by BCR/ABL are highly resistant to a variety of apoptotic stimuli. Resistance to drug-induced apoptosis in CD34+ CML patient cells and BCR/ABL+ cell lines is mediated by downstream signaling pathways activated by BCR/ABL. The signal transducer and activator of transcription 5 pathway which regulates the expression of the anti-apoptotic Bcl-x gene is constitutively activated in BCR/ABL-positive cells. Several lines of evidence led to the hypothesis that Bcl-x is a critical mediator in evasion of apoptosis required to sustain leukemia. In order to examine the in vivo role of Bcl-x in CML we generated a tetracycline-inducible transgenic mouse model in which BCR/ABL ...