Diana Munoz - Academia.edu (original) (raw)
Papers by Diana Munoz
Molecular and Cellular Biology/Genetics
eLife, Feb 1, 2017
Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (M... more Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs(1-4). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of KEAP1, a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in ...
Cancer discovery, Aug 3, 2016
CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. In this stu... more CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. In this study, we compare the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies by performing parallel deep-coverage shRNA and CRISPR screens targeting 2722 genes across several cancer cell lines. CRISPR-based dropout screens identified more lethal genes compared to RNAi in all five cancer models, indicating that the identification of many cellular dependencies may require full gene inactivation, as induced by CRISPR but not RNAi. However, in two aneuploid cancer models we found that all genes within highly amplified regions, including non-expressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits in CRISPR-based screens. Using a CRISPR tiling array that encompassed all possible sgRNAs against the coding regions of 139 genes, we found that sgRNAs targeting essential domains provide the most robust dro...
Oncotarget, 2011
Glioblastoma multiforme (GBM) is the most common and lethal of human primary central nervous syst... more Glioblastoma multiforme (GBM) is the most common and lethal of human primary central nervous system (CNS) tumors, with a median survival of 14-16 months despite optimal surgery, radiation and chemotherapy. A reason for this dismal prognosis is insufficient understanding of the ontogeny of GBMs, which are highly heterogeneous at a pathological level. This pathological diversity, between and within GBMs as well as varying grades of gliomas, has not been fully explained solely on the grounds of oncogenic stimulus. Interaction with the tumor microenvironment is likely a source of this pathological heterogeneity, as well as the inherent characteristics of the tumor cell of origin. Currently, controversy exists on whether the initial transformed cell is a differentiated astrocyte, progenitor or neural stem cell. Putative cancer stem cells (CSCs), which have features of normal stem cell plus the ability to recapitulate the tumor phenotype in vivo in small numbers, have been identified from...
CNS Cancer, 2009
Robust animal models have come to the forefront of understanding GBM biology and cancer biology i... more Robust animal models have come to the forefront of understanding GBM biology and cancer biology in general. Specifically, genetically engineered murine models or GEMs have provided a great deal of understanding in investigating the role of p21-Ras in GBM. Elevation of Ras activity is a molecular hallmark of GBM and is under intense investigation. Several animal models have been engineered
Translational Neuroscience, 2011
Glioblastoma Multiforme (GBM) is the most malignant and devastating primary brain tumour with a m... more Glioblastoma Multiforme (GBM) is the most malignant and devastating primary brain tumour with a median survival of ∼12–16 months. Although recent large scale sequencing projects have shed considerable light into the complexity of the disease, there remains much to be elucidated in the hopes of generating effective therapeutic strategies. Although these studies investigate the mutations and expression of bulk tumour they have limits with respect to cell of origin and the concept of brain tumour initiating cells (BTIC). Current research has challenged the old paradigm of the stochastic model as recent evidence suggests that a subset of cancer cells within a tumor is responsible for tumor initiation, maintenance, and resistance to therapy. To gain a better understanding of the different compartment of cells that GBM comprise of require careful and elegant experiments. In addition to studying GBM, exploring the role of normal neural stem cells and progenitors cells is essential to parti...
CNS Cancer, 2009
Chapter 38 Inhibition of Ras Signaling for Brain Tumor Therapy Sameer Agnihotri, Diana Munoz, and... more Chapter 38 Inhibition of Ras Signaling for Brain Tumor Therapy Sameer Agnihotri, Diana Munoz, and Abhijit Guha ... Cell Biol., 10: 181205. Guha A, Feldkamp MM, Lau N, Boss G, Pawson A (1997) Proliferation of human malignant astrocytomas is dependent on Ras activation. ...
Proceedings of the National Academy of Sciences, 2013
Neurobiology of Disease, 2011
Highly proliferating cells, normal or transformed, undergo aerobic glycolysis whereby glucose is ... more Highly proliferating cells, normal or transformed, undergo aerobic glycolysis whereby glucose is metabolized to lactate rather than by oxidative metabolism, even in the presence of oxygen. This metabolic adaptation provides a survival advantage and facilitates synthesis of biosynthetic precursors required for continued cellular proliferation. An important mediator of aerobic glycolysis is our demonstration that in malignant gliomas there is over-expression of the glycolytic enzyme hexokinase 2 (HK2), phosphorylating glucose as the first step of the glycolytic pathway. In contrast, normal brain preferentially expresses HK1 and undergoes oxidative glucose metabolism. In this study, we examine whether this switch in HK isoform also occurs in the developing embryo and central nervous system (CNS). Bioinformatic analysis of available microarray data, including that of The Cancer Genome Atlas, demonstrated a ~17% overlap in metabolic-related genes in blastocyst stage embryo and human GBM tissue, including upregulation of HK2 and downregulation of HK1. Quantitative RT-PCR on mouse brains isolated at different embryonic and postnatal development time-points demonstrated HK2 expression was highest in the early embryo, while HK1 expression increased with CNS maturation. The downstream glycolytic enzymes PKM2 and LDHA had similar temporal profiles as HK2. Expression of the HK2 isoform was due in part to epigenetic regulation of HK2. In support, adult normal human brain and the few human GBM cell lines with low HK2 expression had methylation of CpG islands within intron 1 of HK2. In contrast, developing human fetal brain and GBM tissue expressing HK2 demonstrated significantly lower percent methylation. Furthermore, treatment of GBM cells lacking HK2 with 5-aza-2-deoxycytidine restored HK2 transcript expression. Overall, our results demonstrate that proliferative states including the developing embryo and malignant gliomas, which rely on aerobic glycolysis, preferentially express the HK2 isoform, found to be regulated in part epigenetically.
Molecular Cancer Research, 2012
Journal of Experimental Medicine, 2011
GATA4 loss as a result of promoter hypermethylation or somatic mutation promotes growth and chemo... more GATA4 loss as a result of promoter hypermethylation or somatic mutation promotes growth and chemotherapy resistance of human astrocytomas.
Glia, 2013
Gliomas are recognized as a heterogeneous group of neoplasms differing in their location and morp... more Gliomas are recognized as a heterogeneous group of neoplasms differing in their location and morphological features. These differences, between and within varying grades of gliomas, have not been explained solely on the grounds of an oncogenic stimulus. Interactions with the tumor microenvironment as well as inherent characteristics of the cell of origin are likely a source of this heterogeneity. There is an ongoing debate over the cell of origin of gliomas, where some suggest a progenitor, while others argue for a stem cell origin. Thus, it is presumed that neurogenic regions of the brain such as the subventricular zone (SVZ) containing large numbers of neural stem and progenitor populations are more susceptible to transformation. Our studies demonstrate that K-ras(G12D) cooperates with the loss of p53 to induce gliomas from both the SVZ and cortical region, suggesting that cells in the SVZ are not uniquely gliomagenic. Using combinations of doxycycline-inducible K-ras(G12D) and p53 loss, we show that tumors induced by the cooperative actions of these genes remain dependent on active K-ras expression, as deinduction of K-ras(G12D) leads to complete tumor regression despite absence of p53. These results suggest that the interplay between specific combinations of genetic alterations and susceptible cell types, rather than the site of origin, are important determinates of gliomagenesis. Additionally, this model supports the view that, although several genetic events may be necessary to confer traits associated with oncogenic transformation, inactivation of a single oncogenic partner can undermine tumor maintenance, leading to regression and disease remission.
Cellular & Molecular Biology Letters, 2010
A clear association between obesity and heart failure exists and a significant role for leptin, t... more A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observe...
Molecular and Cellular Biology/Genetics
eLife, Feb 1, 2017
Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (M... more Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs(1-4). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of KEAP1, a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in ...
Cancer discovery, Aug 3, 2016
CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. In this stu... more CRISPR/Cas9 has emerged as a powerful new tool to systematically probe gene function. In this study, we compare the performance of CRISPR to RNAi-based loss-of-function screens for the identification of cancer dependencies by performing parallel deep-coverage shRNA and CRISPR screens targeting 2722 genes across several cancer cell lines. CRISPR-based dropout screens identified more lethal genes compared to RNAi in all five cancer models, indicating that the identification of many cellular dependencies may require full gene inactivation, as induced by CRISPR but not RNAi. However, in two aneuploid cancer models we found that all genes within highly amplified regions, including non-expressed genes, scored as lethal by CRISPR, revealing an unanticipated class of false-positive hits in CRISPR-based screens. Using a CRISPR tiling array that encompassed all possible sgRNAs against the coding regions of 139 genes, we found that sgRNAs targeting essential domains provide the most robust dro...
Oncotarget, 2011
Glioblastoma multiforme (GBM) is the most common and lethal of human primary central nervous syst... more Glioblastoma multiforme (GBM) is the most common and lethal of human primary central nervous system (CNS) tumors, with a median survival of 14-16 months despite optimal surgery, radiation and chemotherapy. A reason for this dismal prognosis is insufficient understanding of the ontogeny of GBMs, which are highly heterogeneous at a pathological level. This pathological diversity, between and within GBMs as well as varying grades of gliomas, has not been fully explained solely on the grounds of oncogenic stimulus. Interaction with the tumor microenvironment is likely a source of this pathological heterogeneity, as well as the inherent characteristics of the tumor cell of origin. Currently, controversy exists on whether the initial transformed cell is a differentiated astrocyte, progenitor or neural stem cell. Putative cancer stem cells (CSCs), which have features of normal stem cell plus the ability to recapitulate the tumor phenotype in vivo in small numbers, have been identified from...
CNS Cancer, 2009
Robust animal models have come to the forefront of understanding GBM biology and cancer biology i... more Robust animal models have come to the forefront of understanding GBM biology and cancer biology in general. Specifically, genetically engineered murine models or GEMs have provided a great deal of understanding in investigating the role of p21-Ras in GBM. Elevation of Ras activity is a molecular hallmark of GBM and is under intense investigation. Several animal models have been engineered
Translational Neuroscience, 2011
Glioblastoma Multiforme (GBM) is the most malignant and devastating primary brain tumour with a m... more Glioblastoma Multiforme (GBM) is the most malignant and devastating primary brain tumour with a median survival of ∼12–16 months. Although recent large scale sequencing projects have shed considerable light into the complexity of the disease, there remains much to be elucidated in the hopes of generating effective therapeutic strategies. Although these studies investigate the mutations and expression of bulk tumour they have limits with respect to cell of origin and the concept of brain tumour initiating cells (BTIC). Current research has challenged the old paradigm of the stochastic model as recent evidence suggests that a subset of cancer cells within a tumor is responsible for tumor initiation, maintenance, and resistance to therapy. To gain a better understanding of the different compartment of cells that GBM comprise of require careful and elegant experiments. In addition to studying GBM, exploring the role of normal neural stem cells and progenitors cells is essential to parti...
CNS Cancer, 2009
Chapter 38 Inhibition of Ras Signaling for Brain Tumor Therapy Sameer Agnihotri, Diana Munoz, and... more Chapter 38 Inhibition of Ras Signaling for Brain Tumor Therapy Sameer Agnihotri, Diana Munoz, and Abhijit Guha ... Cell Biol., 10: 181205. Guha A, Feldkamp MM, Lau N, Boss G, Pawson A (1997) Proliferation of human malignant astrocytomas is dependent on Ras activation. ...
Proceedings of the National Academy of Sciences, 2013
Neurobiology of Disease, 2011
Highly proliferating cells, normal or transformed, undergo aerobic glycolysis whereby glucose is ... more Highly proliferating cells, normal or transformed, undergo aerobic glycolysis whereby glucose is metabolized to lactate rather than by oxidative metabolism, even in the presence of oxygen. This metabolic adaptation provides a survival advantage and facilitates synthesis of biosynthetic precursors required for continued cellular proliferation. An important mediator of aerobic glycolysis is our demonstration that in malignant gliomas there is over-expression of the glycolytic enzyme hexokinase 2 (HK2), phosphorylating glucose as the first step of the glycolytic pathway. In contrast, normal brain preferentially expresses HK1 and undergoes oxidative glucose metabolism. In this study, we examine whether this switch in HK isoform also occurs in the developing embryo and central nervous system (CNS). Bioinformatic analysis of available microarray data, including that of The Cancer Genome Atlas, demonstrated a ~17% overlap in metabolic-related genes in blastocyst stage embryo and human GBM tissue, including upregulation of HK2 and downregulation of HK1. Quantitative RT-PCR on mouse brains isolated at different embryonic and postnatal development time-points demonstrated HK2 expression was highest in the early embryo, while HK1 expression increased with CNS maturation. The downstream glycolytic enzymes PKM2 and LDHA had similar temporal profiles as HK2. Expression of the HK2 isoform was due in part to epigenetic regulation of HK2. In support, adult normal human brain and the few human GBM cell lines with low HK2 expression had methylation of CpG islands within intron 1 of HK2. In contrast, developing human fetal brain and GBM tissue expressing HK2 demonstrated significantly lower percent methylation. Furthermore, treatment of GBM cells lacking HK2 with 5-aza-2-deoxycytidine restored HK2 transcript expression. Overall, our results demonstrate that proliferative states including the developing embryo and malignant gliomas, which rely on aerobic glycolysis, preferentially express the HK2 isoform, found to be regulated in part epigenetically.
Molecular Cancer Research, 2012
Journal of Experimental Medicine, 2011
GATA4 loss as a result of promoter hypermethylation or somatic mutation promotes growth and chemo... more GATA4 loss as a result of promoter hypermethylation or somatic mutation promotes growth and chemotherapy resistance of human astrocytomas.
Glia, 2013
Gliomas are recognized as a heterogeneous group of neoplasms differing in their location and morp... more Gliomas are recognized as a heterogeneous group of neoplasms differing in their location and morphological features. These differences, between and within varying grades of gliomas, have not been explained solely on the grounds of an oncogenic stimulus. Interactions with the tumor microenvironment as well as inherent characteristics of the cell of origin are likely a source of this heterogeneity. There is an ongoing debate over the cell of origin of gliomas, where some suggest a progenitor, while others argue for a stem cell origin. Thus, it is presumed that neurogenic regions of the brain such as the subventricular zone (SVZ) containing large numbers of neural stem and progenitor populations are more susceptible to transformation. Our studies demonstrate that K-ras(G12D) cooperates with the loss of p53 to induce gliomas from both the SVZ and cortical region, suggesting that cells in the SVZ are not uniquely gliomagenic. Using combinations of doxycycline-inducible K-ras(G12D) and p53 loss, we show that tumors induced by the cooperative actions of these genes remain dependent on active K-ras expression, as deinduction of K-ras(G12D) leads to complete tumor regression despite absence of p53. These results suggest that the interplay between specific combinations of genetic alterations and susceptible cell types, rather than the site of origin, are important determinates of gliomagenesis. Additionally, this model supports the view that, although several genetic events may be necessary to confer traits associated with oncogenic transformation, inactivation of a single oncogenic partner can undermine tumor maintenance, leading to regression and disease remission.
Cellular & Molecular Biology Letters, 2010
A clear association between obesity and heart failure exists and a significant role for leptin, t... more A clear association between obesity and heart failure exists and a significant role for leptin, the product of the obese gene, has been suggested. One aspect of myocardial remodeling which characterizes heart failure is a disruption in the balance of extracellular matrix synthesis and degradation. Here we investigated the effects of leptin on matrix metalloproteinase (MMP) activity, tissue inhibitor of metalloproteinase (TIMP) expression, as well as collagen synthesis in HL-1 cardiac muscle cells. Gelatin zymographic analysis of MMP activity in conditioned media showed that leptin enhanced MMP-2 activity in a dose- and time-dependent manner. Leptin is known to stimulate phosphorylation of p38 MAPK in cardiac cells and utilization of the p38 MAPK inhibitor, SB203580, demonstrated that this kinase also plays a role in regulating several extracellular matrix components, such that inhibition of p38 MAPK signaling prevented the leptin-induced increase in MMP-2 activation. We also observe...