1993 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mutational and Functional Analysis of Wilms'Tumor @ Massachusetts General Hospital
Wilms' tumor is an embryonal malignancy of the kidney which has been linked to the inactivation of one or more tumor suppressor genes. Initial clinical observations noted the relatively frequent occurrence of bilateral tumors, implying an underlying genetic susceptibility in some individuals, as well as the existence of rare congenital syndromes that were associated with Wilms' tumor and displayed specific germline chromosome abnormalities. Molecular studies of sporadic Wilms' tumors demonstrated allelic losses involving two distinct loci on chromosome 11, at bands p13 and p15. Using positional cloning techniques, we were involved in the initial isolation of the 11p13 Wilms' tumor gene, which we have called WT1. WT1 encodes a zinc finger protein with significant homology to the Early Growth Response genes (EGR). It is normally expressed in specific cells within the kidney, during a narrow window in development. Four alternatively spliced mRNA transcripts are produced, and one of these has been shown to recognize a DNA sequence similar to the EGR1 consensus and to mediate transcriptional repression in vitro. We have shown that most Wilms' tumors express high levels of WT1, consistent with their tissue of origin, but that a fraction (10%) contain inactivating mutations within the coding sequence. While most of these mutations result in homozygous inactivation of the gene, some Wilms' tumors contain only a single mutated WT1 allele, retaining the second wild type copy. We have recently shown that such a mutated WT1 allele is capable of exerting a "dominant negative" effect, cooperating with the viral gene E1A in transforming primary kidney cells. We propose to build on our initial observations, focusing on the use of mutational and functional analyses to define the role of WT1 and the types of tumors in which its inactivation contributes to tumorigenesis. We have recently completed the genomic map of WT1, which enables us to PCR amplify exons of the gene from a variety of clinical specimens, and search for the presence of inactivating mutations. We have already shown that WT1 mutations are not limited to Wilms' tumors, but are also found in adult mesotheliomas, asbestos-induced tumors of the pleural lining, a tissue that normally expresses WT1 early in development. We have recently identified Wilms' tumor cell lines bearing WT1 mutations, making it possible to re-introduce the different splice forms of wild type WT1 and test their ability to reverse the transformed phenotype. Finally, we will extend our study of "dominant negative" WT1 mutations, by using these to suppress normal WT1 function in model systems including in vitro models of cellular differentiation and transgenic mice. Insofar as dominant negative WT1 mutations result from disruption of the DNA binding domain, their effect may be mediated by protein interactions, which we will be able to study using the stable transfectant cell lines that we have developed.
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0.907 |
1994 — 1997 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mutational and Functional Analysis of Wilm's Tumor @ Massachusetts General Hospital
Wilms' tumor is an embryonal malignancy of the kidney which has been linked to the inactivation of one or more tumor suppressor genes. Initial clinical observations noted the relatively frequent occurrence of bilateral tumors, implying an underlying genetic susceptibility in some individuals, as well as the existence of rare congenital syndromes that were associated with Wilms' tumor and displayed specific germline chromosome abnormalities. Molecular studies of sporadic Wilms' tumors demonstrated allelic losses involving two distinct loci on chromosome 11, at bands p13 and p15. Using positional cloning techniques, we were involved in the initial isolation of the 11p13 Wilms' tumor gene, which we have called WT1. WT1 encodes a zinc finger protein with significant homology to the Early Growth Response genes (EGR). It is normally expressed in specific cells within the kidney, during a narrow window in development. Four alternatively spliced mRNA transcripts are produced, and one of these has been shown to recognize a DNA sequence similar to the EGR1 consensus and to mediate transcriptional repression in vitro. We have shown that most Wilms' tumors express high levels of WT1, consistent with their tissue of origin, but that a fraction (10%) contain inactivating mutations within the coding sequence. While most of these mutations result in homozygous inactivation of the gene, some Wilms' tumors contain only a single mutated WT1 allele, retaining the second wild type copy. We have recently shown that such a mutated WT1 allele is capable of exerting a "dominant negative" effect, cooperating with the viral gene E1A in transforming primary kidney cells. We propose to build on our initial observations, focusing on the use of mutational and functional analyses to define the role of WT1 and the types of tumors in which its inactivation contributes to tumorigenesis. We have recently completed the genomic map of WT1, which enables us to PCR amplify exons of the gene from a variety of clinical specimens, and search for the presence of inactivating mutations. We have already shown that WT1 mutations are not limited to Wilms' tumors, but are also found in adult mesotheliomas, asbestos-induced tumors of the pleural lining, a tissue that normally expresses WT1 early in development. We have recently identified Wilms' tumor cell lines bearing WT1 mutations, making it possible to re-introduce the different splice forms of wild type WT1 and test their ability to reverse the transformed phenotype. Finally, we will extend our study of "dominant negative" WT1 mutations, by using these to suppress normal WT1 function in model systems including in vitro models of cellular differentiation and transgenic mice. Insofar as dominant negative WT1 mutations result from disruption of the DNA binding domain, their effect may be mediated by protein interactions, which we will be able to study using the stable transfectant cell lines that we have developed.
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0.907 |
1995 — 1998 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Isolation of Gene For X-Linked Lymphoproliferation @ Massachusetts General Hospital
X-linked lymphoproliferative syndrome (XLP) is an inherited immunodeficiency specific to infection by Epstein-Barr virus (EBV). Rather than self-limited infectious mononucleosis following primary exposure to EBV, affected boys develop uncontrolled proliferation of EBV-transformed B cells, often culminating in fatal lymphoma. XLP is therefore a genetic model for the EBV-induced lymphomas seen in MDS patients and in organ transplant recipients. The nature of the inherited defect in XLP patients is unknown, and no consistent immunological abnormalities have been demonstrated in these children prior to EBV infection. We propose experiments aimed at isolating the XLP disease gene by a "positional cloning" approach. The XLP gene has been mapped to a genetic locus at chromosome Xq25, and three unrelated patients with homozygous germline deletions of approximately 2 megabases at that locus have been reported. Boys who are homozygous for this large deletion on the X chromosome have no clinical abnormalities other than XLP, suggesting that no other "critical" genes are present within that chromosomal locus. We identified the first of three known genomic markers within the common region deleted-in all three XLP patients, and have used these as starting points in establishing a yeast artificial chromosome (YAC) contig spanning the deletion. To identify potential transcripts within this region, we are using Exon Amplification, a highly sensitive technique developed to "trap" exons from genomic DNA. Potential exons will be used to screen cDNA libraries and will also be useful as markers in ordering the YAC contig. Candidate cDNAs will be screened by sequencing, tissue distribution of expression, and analysis for mutations in XLP patients who do not have gross chromosomal deletions. The identification of the XLP disease gene will allow biochemical and functional experiments aimed at defining its role in the growth control of EBV-transformed lymphoid cells.
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0.907 |
1998 — 2002 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Functional Properties of the Wilms Tumor Gene Wt1 @ Massachusetts General Hospital
DESCRIPTION: (adapted from the investigator's abstract) WT1 is a developmentally regulated kidney zinc finger transcription factor which is inactivated in the germline of children predisposed to Wilm's tumor. Both loss of WT1 and overexpression of WT1 lead to apoptosis of renal stem cells critical to kidney development and function. Although WT1 is known to bind to a GC-rich consensus sequence and to repress the activity of promoters with which it interacts, there remain a paucity of genes identified as physiological WT1 targets. WT1 is alternately spliced but no specific function has been assigned to the different splice forms that are prevalent. Thus, while WT1 is a paradigm for a tissue-specific tumor suppressor, little is known about its function, regulatory pathways, and role in tissue differentiation. Dr. Haber proposes to analyze a candidate target gene he has identified, the cell cycle kinase inhibitor p21, and to identify other potential target genes using an inducible cell system. He also aims to identify WT1-binding proteins by two hybrid and immunological approaches. Two model systems are proposed for use to study the functional interactions with candidate target genes and binding proteins. The first system is based on a kidney stem cell assay and the second on a hematopoietic model in which WT1 appears to contribute to development arrest and leukemogenesis. Finally, Dr. Haber proposes to seek other potential Wilm's tumor genes using his collection of normal/tumor pairs of DNAs and the RDA approach developed by Lisitsyn and Wigler.
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0.907 |
2000 — 2001 |
Haber, Daniel A. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Mutational Analysis of P53 Related Genes @ Massachusetts General Hospital
DESCRIPTION: (Applicant's Description) Germline mutations in p53 account for 60-70 percents of cases of Li-Fraumeni Syndrome (LFS), a familial cancer phenotype associated with breast cancer, sarcomas and other tumors. We hypothesize that LFS cases without p53 mutations may harbor mutations in other components of this DNA damage response pathway. Recent molecular studies have identified kinases that are involved in the phosphorylation and activation of p53, as well as other genes that modulate p53 stability, and its ability to mediate apoptosis. Cellular homologs of p53 have recently been identified, as have mammalian homologs of yeast cell cycle checkpoint genes that are critical to the DNA damage response pathway. The small size of LFS families makes classical linkage analysis difficult, and we therefore propose a candidate gene approach to identify the additional gene (s) responsible for LFS, using high throughput mutational detection techniques. We note that such gene (s) may be involved in both genetic predisposition to cancer and somatic tumorigenesis, but as for BRCAl and BRCA2, absence of somatic mutations does not exclude the possibility of gennline mutations, requiring mutational analysis both in germline and tumor specimens. We propose a phased approach: initially we will undertake mutational analysis in a highly selected cohort of patients with LFS or variant multi-cancer syndromes that do not have mutations in p53. The absence of p53 mutations in such families enhances the likelihood of detecting mutations in related genes required for genomic stability. As a second step, we will expand the mutational analysis to subsets of the general population at high risk for breast cancer, but without the extraordinary risk factors demonstrated by LFS and related families. Finally, we will examine a collection of tumor cell lines for the frequency of mutations in these candidate genes.
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0.907 |
2000 — 2004 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Functional Characterization of Brca1 @ Massachusetts General Hospital
DESCRIPTION: (Adapted from the investigator's abstract) Germline mutations in BRCA1 account for 50% of familial breast cancer and 5-10% of breast cancer cases with an early age of onset. BRCA1 encodes a novel protein implicated in the cellular response to DNA damage, with possible roles in homologous recombination, as well as transcriptional regulation. As an initial step to understanding its functional properties, we have established cells with tightly regulated inducible expression of full length BRCA1, and have identified downstream targets using a microarrayed oligonucleotide screening strategy. We have observed that overexpression of BRCA1 induces expression of the DNA-damage responsive gene GADD45, which in turn activates JNK/SAPK-dependent apoptosis. In this proposal, we will expand on these BRCA1 overexpression studies and develop a loss-of-function model. First, we will use cells with inducible expression of a tagged BRCA1 construct to investigate the post-translational changes in BRCA1 that occur following DNA damage, including phosphorylation, and alterations in size of the in vivo BRCA1 complex and associated proteins. Having identified GADD45 as a BRCA-1 target gene, we will analyze the role of BRCA1 in transcriptional regulation by defining the BRCA1-responsive sequences in the GADD45 promoter, searching for DNA-binding protein(s) that mediate the induction of GADD45 by BRCA1, and characterizing the nature of their interaction with BRCA1 itself. The requirement for GADD45 to mediate the effects of BRCA1 will be tested using a previously defined antisense strategy. Second, we will develop dominant negative constructs to investigate BRCA1-dependent DNA damage response pathways and their contribution to mammary tumorigenesis. Our strategy will be to generate fusion proteins containing the protein interaction "ring" domain of BRCA1 or its associated protein BARD1, fused to a heterologous domain leading to altered subnuclear localization. The effectiveness of these dominant negative constructs will be assayed by immunofluorescence and immunoprecipitation studies. Successful dominant negatives will be expressed in cell lines and in primary cells to identify DNA damage responsive genes whose expression is dependent upon BRCA1 function. To analyze the consequences of BRCA1 inactivation in developing mammary tissue, transgenic mice will be generated using MMTV-driven dominant negative constructs. By analysis of altered gene expression profiles following BRCA1 overexpression, together with the disruption of BRCA1 function using dominant negative constructs, we expect to gain insight into the function of this tumor suppressor.
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0.907 |
2002 — 2006 |
Haber, Daniel A. |
P01Activity Code Description: For the support of a broadly based, multidisciplinary, often long-term research program which has a specific major objective or a basic theme. A program project generally involves the organized efforts of relatively large groups, members of which are conducting research projects designed to elucidate the various aspects or components of this objective. Each research project is usually under the leadership of an established investigator. The grant can provide support for certain basic resources used by these groups in the program, including clinical components, the sharing of which facilitates the total research effort. A program project is directed toward a range of problems having a central research focus, in contrast to the usually narrower thrust of the traditional research project. Each project supported through this mechanism should contribute or be directly related to the common theme of the total research effort. These scientifically meritorious projects should demonstrate an essential element of unity and interdependence, i.e., a system of research activities and projects directed toward a well-defined research program goal. |
Genetic Models of Cellular Proliferation @ Massachusetts General Hospital
DESCRIPTION (provided by applicant): The advent of comprehensive genomic resources has led to the rapid identification of novel genes with potential roles in regulating cellular proliferation, but only limited approaches to define their role within functional pathways and their contribution to human cancer. Genome projects in multiple organisms have underscored the evolutionary conservation of cellular proliferation pathways between man and simpler organisms, and it is the goal of this P01 application to provide a bridge between studies in genetic model organisms and human cancer. The use of screens for novel genes involved in cell proliferation and cell cycle regulation based in Drosophila and C. elegans will lead to the discovery of mammalian orthologs, whose role in human cancer will be assessed by mutational studies. C. elegans and Drosophila also provide efficient tools, such as RNA interference (RNAi), for the functional analysis of novel genes identified in these screens and in mammalian genomic screens targeting homozygous deletions in mouse tumor models. Project 1 (Dyson): Identify novel modifiers of the cell cycle regulators E2F and retinoblastoma (RB) in Drosophila, isolate their mammalian orthologs, and search for mutations in human cancers. Project 2 (Hariharan): Use a recombination-driven screen in the Drosophila eye for genes whose homozygous inactivation triggers increased cellular proliferation, characterize promising candidates, including a novel gene Salvador, and determine whether it is targeted by mutations in human tumors. Project 3 (Haber): Adapt Representational Difference Analysis (RDA) to screen for homozygous genomic deletions in a mouse tumor model of cancer progression, characterize DOS, a novel gene implicated in Rho signaling that is targeted by such a deletion, use RNAi to analyze novel genes present within tumor-associated deletions. Project 4 (van den Heuvel): Use C. elegans to search for genes that modulate the function of D-type cyclins, characterize two novel negative regulators, lin-9 and lin-36, and define their contribution to human cancer. These projects will be supported by cores for Administration (Haber), Genetics (Drosophila: Artavanis-Tsakonas, and C. elegans: Hart), and Molecular Pathology (expression: Stamenkovic, and mutational analysis: Bell). Collectively, these studies provide a concerted effort to make use of powerful tools provided by genetic model systems to define the function of new genes involved in cellular proliferation and their potential roles in human cancer.
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0.907 |
2002 — 2006 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Targets of Ewing Sarcoma-Wilms Tumor 1 Oncoprotein @ Massachusetts General Hospital
Desmoplastic Small Round Cell Tumor (DSRCT) is an embryonal cancer affecting young adults, which is thought to originate in primitive cells of mesothelial origin. The defining histology of DSRCT consists of islets of tumor cells surrounded by reactive fibrosis (desmoplasia), suggesting that tumor/stromal interactions play an important role in the genesis of this cancer. The defining genetic characteristic of DSRCT is a consistent chromosomal rearrangement, fusing the N-terminal transactivational domain (NTD) of the Ewing Sarcoma gene EWS to the three C-terminal zinc fingers of the Wilms tumor suppressor gene WT1, whose DNA binding domain is modulated by alternative splicing (plus/minus insertion of three amino acids, KTS). This complex chimeric transcriptional activator constitutes a single oncogenic gain-of-function mutation that underlies malignant transformation in DSRCT. Identification and characterization of physiologically-relevant target genes of EWS-WT1 will therefore provide important insight into the mechanism of tumorigenesis, and specifically into the interaction between epithelial cancer cells and surrounding stroma, for which DSRCT constitutes a striking model. We have generated osteosarcoma cells with inducible expression of EWS-WT1(plus/minus KTS), and used hybridization to high density microarrays, as well as subtractive PCR, to identify endogenous transcripts whose expression is regulated by EWS-WT1. Extensive validation of these potential targets revealed two novel genes that are directly induced by EWS-WT1 and are expressed in primary DSRCT tumor specimens. We propose to characterize MLF1, a recently identified gene implicated in lineage switching and apoptosis, which is specifically induced by the EWS-WT1(-KTS) isoform, and the novel gene, LRT1, which encodes a novel transmembrane type I glycoprotein that is highly induced by EWS-WT1(+KTS). The mechanism(s) by which these genes are regulated by EWS- WT1(plus/minus kTS), their functional properties and their potential contribution to EWS-WT1-mediated transformation will be studied. Mouse xenograft and cell line models for EWS-WT1- dependent transformation will be established to define the relative contributions of these genes, along with those of two previously characterized EWS-WT1(-KTS) targets, Platelet-Derived Growth Factor-A (PDGF-A) and IL2-receptor beta. Understanding the contribution of target genes regulated by the oncogenic EWS- WT1 chimeric transcription factor will provide important insight into this cancer model, which exemplifies the interactions between tumor cells and their surrounding stroma.
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0.907 |
2003 — 2007 |
Haber, Daniel A. |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Functional Properties of the Wilm's Tumor Gene Wt1 @ Massachusetts General Hospital
DESCRIPTION (provided by applicant): The genetic pathways that underlie the development of the pediatric kidney cancer Wilms tumor provide unique insight into the link between normal development of the kidney and its deregulation during malignant transformation. Inactivating mutations in the WT1 tumor suppressor, a zinc finger transcription factor encoded by multiple alternative splicing variants, underlie a subset of Wilms tumors, pointing to critical transcriptional targets that contribute to kidney development and tumorigenesis. We have used cells with inducible expression of the transcription allyactive isoform, WT1 (-KTS), combined with expression profile analysis to identify physiologically regulated target genes, and we have established a model for WT1-directed cellular differentiation using hematopoietic precursors. Here, we propose to focus on the functional properties of the most abundant isoform, WT1 (+KTS), whose function is unknown. In Aim I, we will search for endogenous genes whose expression is regulated by WT1 (+KTS), using both hybridization to microarrays and subtractivePCR approaches. The mechanisms by which this WT1 isoform regulates expression of its target genes (transcriptional as well as postulated post-transcriptional mechanisms) will be studied. The functional properties of these downstream effectors and their potential contribution to renal differentiation and Wilms tumorigenesis will be addressed. In Aim II, we will pursue protein-protein interactions that have been implicated in WT1 (+KTS) function, as well as in its characteristic subnuclear localization within "speckles". A combination of yeast-two hybrid assays and mass spectrometry sequencing of coprecipitated proteins will be employed, and the functional significance of confirmed interactors will be addressed. In Aim III, we will study another recently isolated transcription factor, BF2, which is also essential for kidney development, but through a distinct mechanism. Remarkably, BF2 is expressed only in stromal cells of the fetal kidney, yet its inactivation in the mouse suppresses epithelial differentiation without affecting stromal cells themselves. Using inducible expression of BF2 and expression profile analysis, we have identified downstream targets, including secreted growth factors, whose contribution to renal differentiation and to stromal-epithelial interactions will be explored. Taken together, this proposal aims at identifying downstream targets of two transcription factors that are essential to kidney development, defining the mechanism by which these targets are regulated, and studying their potential contribution to Wilms tumorigenesis.
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0.907 |
2008 — 2012 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Circumventing Acquired Resistance to Growth Factor Receptor Kinase Inhibitors @ Massachusetts General Hospital
DESCRIPTION (provided by applicant): The success of molecularly targeted cancer therapy using tyrosine kinase inhibitors (TKIs) faces a number of difficult challenges. Foremost among these is the ability to identify subsets of different cancers that are uniquely sensitive to targeted agents, often identified by the presence of genetic markers implying dependence or addiction to the targeted pathway. Equally important to the longterm success of these therapies is understanding and circumventing acquired drug resistance, which is a key limitation to their clinical effectiveness. Acquired resistance to drugs targeting growth factor receptors differs from resistance to genotoxic cancer chemotherapy, and may include both specific mutations in targeted receptors, as well as more complex functional alterations in signaling networks. Here we will use non-small cell lung cancer (NSCLC) cell line models that appear to faithfully recapitulate key signaling dependence of cancers with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene, identifying a subset of lung cancers with extreme sensitivity to EGFR TKIs. We outline three aims that address the acquisition of resistance in tumors that were previously sensitive to these agents: in Aim 1, we will generate cell line models for acquired resistance to second generation irreversible inhibitors of EGFR, and use genetic, signaling and functional analyses to dissect the underlying mechanisms. In Aim 2, we will use a high throughput shRNA screen of tyrosine kinases to identify candidate targets whose suppression may circumvent resistance to EGFR inhibitors. In Aim 3, we will use lentiviral knockdown/reconstitution experiments to quantitate oncogene dependence of drug resistant cells, both on the initiating EGFR mutation and on associated signaling pathways that contribute to acquired drug resistance. Together, these aims will provide important insight into critical mechanisms that underlie the acquisition of resistance to novel inhibitors targeting growth factor receptors in human cancer. PUBLIC HEALTH RELEVANCE: Understanding the mechanisms by which cancers that are sensitive to the new classes of targeted cancer therapies become resistant to these is critical to their eventual clinical success. Our approach is designed to dissect the molecular basis of resistance to these cancer drugs.
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0.907 |
2008 — 2011 |
Haber, Daniel A. |
R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Functional Properties of the Wilms'Tumor Gene Wt1 @ Massachusetts General Hospital
Wilms tumor (nephroblastoma) is a pediatric kidney cancer that arises in renal precursor cells and constitutes a prime example of the link between normal organ development and tumorigenesis. The WT1 tumor suppressor gene encodes a zinc finger transcription factor, thought to be a master regulator of kidney differentiation, whose inactivation in a subset of Wilms tumors triggers malignant proliferation. We have characterized the two primary WT1 splicing variants (+/-KTS): WT1(-KTS) encodes a transcriptional regulator of a cellular differentiation program, but the function of the equally essential WT1(+KTS) isoform is unknown. Using a chromatin immunoprecipitation strategy, we have recently uncovered potential targets of WT1(+KTS) and now propose to characterize their regulation by WT1 and their role in normal renal differentiation and tumorigenesis. We have also studied additional transcriptional regulators that affect renal differentiation, including the Forkhead transcription factor BF2/FoxD, whose expression by stromal cells is required for the differentiation of neighboring epithelial cells, identifying the secreted Placenta! Growth Factor as a transcriptional target of BF2. We have also studied Polycomb group genes (PcG), which are essential for maintaining the repressed state of Hox genes, using the single C. elegans ortholog SOP-2 to define key functional elements that are highly conserved across evolution. To search for additional Wilms tumor genes, we have used array comparative genomic hybridization to identify novel sites of chromsomal gains and losses in primary Wilms tumor specimens. We have identified two novel candidate genes that are targeted by intragenic mutations in a significant number of cases. We propose genetic and functional analyses of these new candidate Wilms tumor genes. Relevance of Research Project to Public Health: Wilms tumor is the most common kidney cancer in children, and while many cases are now curable, a significant number of children still die of this tumor. An understanding of the genetic causes of Wilms tumor is now possible and may lead to improved classification and subtype-specific treatment for differnt forms of this cancer. In addition, Wilms tumor is an invaluable model for other embryonal tumors of childhood, and defining the relevant mechanisms that govern its proliferation will have important impact for other human tumors that originate in such precursor cells.
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0.907 |
2009 |
Haber, Daniel A. |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
Clinical Correlations of Wtx Inactivation in Wilms Tumor @ Beth Israel Deaconess Medical Center
Stale the application's broad, long-term objectives and specific aims, making reference to ttie health relatedness of the project (i.e., relevance to the mission of the agency). Describe concisely the research design and methods for achieving these goals. Describe the rationale and techniques you will use to pursue these goals. In addition, in two or three sentences, describe in plain, lay language the relevance of this research to public health. If the application is funded, this description, as is, will become public infonnation. Therefore, do not include proprietary/confidential infonnation. DO NOT EXCEED THE SPACE PROVIDEO Wilms tumor is the most common pediatric kidney cancer and is closely connected to kidney development. Mutafions in two genes, WT1 and beta-catenin, and epigenefic changes in the insulin-like growth factor 2 (IGF2) locus have been described but the genetic basis of the majority of cases remains unknown. Given that current treatment protocols for Wilms tumor achieve high success rates (85%), there is a pressing need for prognosfic markers that can guide clinical management but these have been difficult to define. We have recently identified a novel tumor suppressor located on the X chromosome, WTX, which is inactivated in 30% of Wilms tumor cases. We now propose to build on our initial findings to establish clinical correlates of WTX inacfivafion and to test the potenfial applicafion of WTX mutafions as markers of prognosis. We wil also define additional markers by identifying molecular pathways that are affected by WTX inactivation Together, these studies will achieve immediate translational goals by defining novel biomarkers in Wilms tumor and will further our understanding of this disease to allow the future development of biologically based therapies. Specific aims: 1) To define clinical correlafions of WTX inactivation in Wilms tumor. We will analyze 200 Wilms tumors for WTX mutations and correlate our findings with disease outcomes and other clinical parameters such as age at presentation, stage at diagnosis, bilaterality and associated developmental malformafions. We will also develop a WTX polyclonal antibody and a Wilms tumor fissue microarray to test WTX protein levels. 2) Modeling WTX funcfion to identify pathways of potential clinical significance. We will use immunoprecipitation of tagged WTX followed by mass spectrometry to define interactions between WTX and other cellular components. The funcfional consequences of these interactions will be validated in vitro using kidney derived cell lines and in vivo using a WTX conditional knockout mouse. 3) Clinical validation of novel Wilms tumor markers. Genes involved in WTX related pathways will be tested for potenfial clinical applicafions as biomarkers by correlafing expression levels with clinical parameters. We will also test selected genes for mutafions in primary Wilms tumors. We anticipated that, by defining prognostic markers and furthering our knowledge of WTX related pathways in Wilms tumor, this project will have public health applications in the treatment of pediatric kidney cancer.
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0.922 |
2010 — 2013 |
Haber, Daniel A. |
P50Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These grants differ from program project grants in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes. |
P1 - Clinical Correlations of Wtx Inactivation in Wilms Tumor @ Beth Israel Deaconess Medical Center
Wilms tumor is the most common pediatric kidney cancer and is closely connected to kidney development. Mutafions in two genes, WT1 and beta-catenin, and epigenefic changes in the insulin-like growth factor 2 (IGF2) locus have been described but the genetic basis of the majority of cases remains unknown. Given that current treatment protocols for Wilms tumor achieve high success rates (85%), there is a pressing need for prognosfic markers that can guide clinical management but these have been difficult to define. We have recently identified a novel tumor suppressor located on the X chromosome, WTX, which is inactivated in 30% of Wilms tumor cases. We now propose to build on our initial findings to establish clinical correlates of WTX inacfivafion and to test the potenfial applicafion of WTX mutafions as markers of prognosis. We wil also define additional markers by identifying molecular pathways that are affected by WTX inactivation Together, these studies will achieve immediate translational goals by defining novel biomarkers in Wilms tumor and will further our understanding of this disease to allow the future development of biologically based therapies. Specific aims: 1) To define clinical correlafions of WTX inactivation in Wilms tumor. We will analyze 200 Wilms tumors for WTX mutations and correlate our findings with disease outcomes and other clinical parameters such as age at presentation, stage at diagnosis, bilaterality and associated developmental malformafions. We will also develop a WTX polyclonal antibody and a Wilms tumor fissue microarray to test WTX protein levels. 2) Modeling WTX funcfion to identify pathways of potential clinical significance. We will use immunoprecipitation of tagged WTX followed by mass spectrometry to define interactions between WTX and other cellular components. The funcfional consequences of these interactions will be validated in vitro using kidney derived cell lines and in vivo using a WTX conditional knockout mouse. 3) Clinical validation of novel Wilms tumor markers. Genes involved in WTX related pathways will be tested for potenfial clinical applicafions as biomarkers by correlafing expression levels with clinical parameters. We will also test selected genes for mutafions in primary Wilms tumors. We anticipated that, by defining prognostic markers and furthering our knowledge of WTX related pathways in Wilms tumor, this project will have public health applications in the treatment of pediatric kidney cancer.
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0.922 |
2010 — 2012 |
Haber, Daniel A. |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Data Production, and Informatics and Integration
This proposal will create a center for Large Scale Production of Perturbagen-lnduced Cellular Signatures at Harvard Medical School and collaborating institutions, with a focus on perturbations provoked by small molecule drugs and cellular signatures measured using diverse biochemical and single-cell assays. The result will be a large, self-consistent and diverse set of network-centric Pharmacological Response Signatures that provide unique insight into disease processes, drug mechanism/selectivity and ultimately patient-specific responses to therapy. The initial focus of the Center will be small molecule kinase inhibitors, versatile perturbagens with high translational potential. We will use known inhibitors and also expand dramatically the publicly documented collection of inhibitors through new medicinal chemistry and use of kinome-wide selectivity assays. The responses of a large collection of human tumor cells and some primary cells to kinase inhibitors, will be assayed using multiplex biochemical assays (for 20-100 proteins) involving bead-based sandwich immunoassays and reverse-phase lysate microarrays, and single-cell assays (using imaging and flow cytometry) for cell cycle state, commitment to senescence or apoptosis, mesenchymal vs. epithelial phenotype and markers of primitive (stem-cell) status. Data will be collected, integrated and distributed using a series of novel, interoperable software tools that manipulate semantically-typed data arrays based on a new XML/HDF5 format. A multi-faceted informatics program will link these phenotypic and biochemical measures of cellular response to a rich and growing set of genomic data being collected by others. These goals will be met through pursuit of six linked specific aims. Aim 1 will focus on existing - largely clinical grade - kinase inhibitors and a set of 45 cell lines that are known to display diverse drug responses land for which extensive genomic data are available. Aim 2 will enlarge the set of perturbagens by developing a large library of kinase inhibitors using new and existing chemistry and profiling biochemical specificity across the kinome. Aim 3 will combine existing and novel compounds in a dose-response analysis across a set of >1000 tumor cell lines to identify representative cell lines and outliers which, in Aim 4, will subjected to detailed analysis at a single-cell level. Aims 5-6 will develop and deploy the information processing systems needed to collect, systematize and distribute diverse data types. This will involve a novel set of interoperable software tools that incorporate emerging no-SQL and semantic web concepts. Methods for adaptive experimental design will be developed to focus data collection on those areas of the doseresponse landscape where signatures are most informative. The final product will be a large publiclyavailable data set radically different from, but highly complementary to, the expression profiles and genome data that are the primary focus of current high-throughput biological studies on perturbagen-lnduced cellular signatures.
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1 |
2013 — 2017 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Modeling and Circumventing Emt to Suppress Metastasis @ Massachusetts General Hospital
DESCRIPTION (provided by applicant): The majority of cancer deaths are caused by blood-borne metastasis from a primary epithelial tumor, but our understanding of this process is limited. Epithelial-to-Mesenchymal Transition (EMT) is a fundamental developmentally regulated change in cell fate, whose aberrant activation in cancer has been proposed as contributing to the invasiveness and motility of cancer cells. However, given the difficulty in studying human cancer metastasis, most studies have relied on cell line and mouse models, and the relevance of EMT in human cancer is not well established. New technologies enabling the isolation and molecular analysis of circulating tumor cells (CTCs), rare cancer cells that are in transit through the bloodstream, now provide a unique opportunity to define mechanisms involved in human cancer metastasis. We propose a molecular analysis of CTCs to validate the prevalence of EMT in human breast cancer, use a carefully titrated in vitro model of EMT to identify key effectors that could constitute potential drug targets, and test their effectiveness using a mouse model in which CTC quantitation provides a rapid readout for the metastatic potential of human breast cancer cells. Our approach has three aims: in Aim 1, we will determine whether EMT is a consistent feature of different histological subtypes of breast cancer and whether these markers evolve dynamically during response or resistance to therapy. This will be accomplished using RNA-in-situ probes that we have developed, capable of scoring quantitative epithelial and mesenchymal markers within individual cells. RNA sequencing will then be applied to identify EMT associated transcripts within breast circulating tumor cells, thus providing insight into the relevant biological pathways. In Aim 2, we will study an inducible in vitro model of EMT, which effectively mediates a regulated epigenetic switch. We have generated timelines of both transcriptionaland chromatin immuno-precipitation profiles, which will be used to identify candidate effectors of EMT, seeking potential drugable targets. In Aim 3, we will establish a robust mouse assay to monitor the ability of breast cancer cells to metastasize and thus test potential suppressors of EMT-related phenotypes. CTC enumeration in the mouse model will be used as a rapid and quantifiable readout for vascular invasiveness. Together, these experiments aim to combine novel molecular and bioengineering technologies to address the relevance of EMT as a therapeutic target in suppressing human cancer metastasis.
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0.907 |
2015 — 2018 |
Haber, Daniel A. Toner, Mehmet |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Point-of Care Microfluidics For Early Detection of Cancer @ Massachusetts General Hospital
? DESCRIPTION (provided by applicant): Early cancer detection is not only critical in providing curative therapies for the vast majority of solid malignancies and preventing mortality, but also reducing morbidity and costs. However, most existing strategies for early detection suffer from poor specificity: they are as likely to cause complications and deaths from false positives or overdiagnosis of non-invasive cancers as they are from identifying true cancers at an early stage. Recent data from our own group and others show that Circulating Tumor Cells (CTCs) may be shed in significant numbers into the blood stream of patients with invasive but localized and early-stage cancers. These observations suggest that, rather than being a rare and late event in the evolution of cancer, the presence of CTCs may be an early herald of tumor vascular invasion, preceding a considerable period of time for the eventual establishment of viable distant metastases. Our specific strategy is to develop a highly sensitive digital readout based on RNA in CTCs using the microfluidic CTC isolation approach (CTC-iChip) combined with digital droplet RNA (ddPCR) measurement platform. It is important to note that the CTC-iChip is unique in that it makes no a priori assumption about the type of the tumor cells and as such it applies to all cancers. Detecting tumor lineage specific RNA within noninvasively accessed tumor cells is the technology most likely to be not only highly specific and sensitive but also universal for early detection of invasive cancer. To this end, we have 3 distinct but interrelated Aims. In Aim I, we will integrate CTC-iChip and digital droplet PCR to develop a highly specific and sensitive RNA-iChip. In Aim II, we will select two cancers for which sensitive but nonspecific screening tests are currently available, prostate and lung cancer, and we propose to transform these into robust and reliable tests using RNA-iChip that would enable broad screening for invasive and curable cancers. In Aim III, we propose to develop the point of care RNA-iChip broad dissemination beyond academic medical centers to local hospitals, oncology clinics and to physicians' offices. Success with prostate and lung cancers would have a profound impact in decreasing cancer morbidity and mortality, and open a path toward broad-based early detection of multiple cancers.
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0.907 |
2018 — 2020 |
Haber, Daniel A. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Modeling Metastasis and Acquired Drug Resistance Using Circulating Tumor Cells @ Massachusetts General Hospital
Project Summary Advanced estrogen receptor (ER)-positive breast cancers are initially responsive to multiple therapeutic interventions, but they ultimately develop drug resistance and disseminate to multiple metastatic sites. Circulating tumor cells (CTCs) underlie the blood-borne spread of cancer and they also provide a noninvasive source to sample, monitor and analyze tumor evolution in real time, as patients develop progressively resistant disease with new metastatic lesions. To enable the detailed molecular study of CTCs, which are extremely rare cells in the circulation, we have made use of microfluidic platforms that efficiently deplete normal hematopoietic cells from blood specimens, leaving behind an enriched population of intact CTCs, some of which remain viable. During the past funding period, we established a panel of patient-derived breast cancer CTC cell lines (Yu et al., Science 2014), which provide a window into critical and poorly understood properties of advanced breast cancer, with significant clinical implications. We demonstrated that these heterogeneous ER+ drug-resistant breast cancer cells contain distinct phenotypes, with a HER2-expressing proliferative state interconverting spontaneously with a Notch1- driven drug resistant state (Jordan et al., Nature 2016). In Aim 1, we will build on this observation to define the likely epigenetic mechanisms that modulate this phenotype conversion. Using live-reporter constructs, we will isolate single cells as they switch between phenotypes to define early transcriptional changes, and in bar-coded pooled knockdown screens, we will test how chromatin modulators affect this phenotype switch, both spontaneously and following the dramatically enhanced reactive oxygen species (ROS)- mediated conversion that we have observed. In Aim 2, we will study another unexpected observation made with cultured breast CTCs, namely their acquired quiescence following direct intravascular inoculation and dissemination to the lung. While a 200 CTC inoculum can initiate tumorigenesis in the mammary gland, tail vein inoculation of 200,000 CTCs leads to non-proliferative single cells throughout the lung, an observation that may be linked to ROS stress experienced by these cells in the bloodstream (Zheng et al., Nature Comm, 2017). We have used pooled bar-coded knockdown construct libraries of chromatin modulators to uncover candidate regulators that are enriched as CTCs eventually initiate proliferation in the lung, and these will be tested individually and in combination, validated in multiple CTC lines, and matched with RNA seq transcriptomes of early stages in the transition from quiescence to early proliferative metastatic lesions. In Aim III, we will examine organ-specific pathways that enable proliferation of breast CTCs in the brain versus bone or liver. By serial inoculation, we have generated derivative lines of CTCs that grow efficiently following direct implantation in brain, compared with parental cells which exhibit a prolonged delay. Using both RNA sequencing and whole proteome mass spectrometry, we will identify modulators of organ-predominant metastasis, which will then be validated through functional assays and correlated with primary CTCs from patients with organ-predominant metastases. All together, these experiments will use patient-derived cultures of metastatic precursors to better understand and ultimately target breast cancer progression.
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0.907 |
2018 — 2021 |
Haber, Daniel A. Maheswaran, Shyamala Toner, Mehmet |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Metastasis and Biophysics of Clusters of Circulating Tumor Cells in the Microcirculation @ Massachusetts General Hospital
ABSTRACT Circulating tumor cells drive metastasis when they travel from primary tumors to distant organs via the circulation. Multicellular clusters of circulating tumor cells though less frequently observed in blood, are much more likely to establish metastases than individual circulating tumor cells and the presence of tumor clusters in blood has been associated with dramatically worse prognoses in patients. Although there are many suspected explanations for their greater metastatic potentials, much is still unknown about the behavior of clusters, especially in the narrow vessels of the body. Recent evidence has demonstrated that cluster transiting through narrow constrictions experience dynamic changes to structure and organization. Forces in the microcirculation cause clusters to reversibly re-organize into single-file chains to enable transit through narrow capillary-sized vessels and nuclear envelopes are ruptured and rapidly repaired during migration events through narrow constrictions. Two biophysical parameters within clusters, cellular adhesion strengths and nuclear mechanics, are vital for these behaviors. Because of the important role that these parameters play in many aspects of metastatic progression, we hypothesize that these parameters modulate the biophysical responses of clusters to physical forces in the microcirculation, and that these interactions play a significant role in the competitive edge that clusters have edge over individual cancer cells for seeding metastases. To this end, we propose three specific aims. In aim 1, we will develop next generation models of the human microcirculation with rounded networks of endothelial cell coated microfluidic devices and geometry matched computational simulations. In aim 2, we will explore how intercellular adhesions affect the biophysical responses and metastasis-forming abilities of homogeneous versus heterogeneous clusters in the microcirculation through the use of our developed models. Finally, in aim 3 we will study the physical basis for nuclear envelope rupture, DNA-damage, genetic instability and other DNA-level affects that are involved in metastatic progression. Understanding the interplay between the biophysics and biology of clusters within the microcirculation will elucidate mechanisms that can be used to combat the progression of cluster-initiated metastases.
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0.907 |
2019 — 2021 |
Haber, Daniel A. Shaw, Alice T |
T32Activity Code Description: To enable institutions to make National Research Service Awards to individuals selected by them for predoctoral and postdoctoral research training in specified shortage areas. |
Training Program in Cancer Biology and Therapeutics @ Massachusetts General Hospital
ABSTRACT The Massachusetts General Hospital (MGH) Cancer Center proposes to renew its T32 Training Program in Cancer Biology and Therapeutics, supporting mentorship of seven physician scientists (MDs and MD/PhDs) for careers in cancer research. With a 20-year history, the program has enrolled multi-disciplinary trainees who have completed clinical fellowships in medical, surgical, pediatric, or radiation oncology, and supported their training in laboratory-based or clinical cancer research. The program aims to prepare the next generation of physician scientists for academic careers in oncology, strengthening fundamental research in cancer biology and its translation into clinical therapeutics. Program co-Directors are Dr. Daniel Haber, an accomplished laboratory-based cancer genetics investigator and Dr. Alice Shaw, a leading clinical researcher in thoracic oncology. They will oversee all administrative aspects of the program, with Dr. Haber overseeing the mentoring of lab-based trainees and Dr. Shaw overseeing the mentoring of clinical research trainees. An Internal Advisory Committee (IAC) will participate in the competitive selection of trainees and in the selection of faculty for the roster of mentors; an External Advisory Committee (EAC) will convene annually to evaluate the strategic direction and success of the program. Mentors are drawn from multiple disciplines and departments, primarily from MGH, and including selected faculty from neighboring MIT and other Harvard institutions. Special emphasis has now been placed on recruiting new mentors in the emerging field of cancer immunology. Trainees are selected across multiple clinical oncology specialities, from programs that are among the most competitive in their respective fields, with a commitment to enhancing diversity. In addition to facilitating the selection of an appropriate research mentor, the T32 program provides both mandatory and optional courses, as well as a broad range of educational experiences. The formal educational offerings have been strengthened with required didactic courses in biostatistics/computational biology and in the ethical conduct of research, along with specialized optional courses. Trainees will now present their work at an annual retreat. PDs and the IAC will be involved in the evaluation of their progress. Success is measured by trainees' academic productivity during and after their T32 support, as well as by their self-reported learning experience. In summary, the MGH T32 Training Program in Cancer Biology and Therapeutics has a long track record of success in providing a rigorous and comprehensive scientific foundation to outstanding physician investigator trainees who aim to establish successful academic research careers in oncology. The program's strength in multi-disciplinary training and integrated laboratory and clinical investigation has been further improved with an enhanced focus and dedicated educational resources, as recommended by the reviewers. With continued funding from NIH, this training program will build on its longstanding success in training future leaders in academic oncology and help advance the field of cancer research globally.
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0.907 |
2021 |
Haber, Daniel A. Maheswaran, Shyamala Toner, Mehmet |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Microfluidic Sorting of Lung Cancer Cells From Leukapheresis Product as An Alternative to Metastatic Tumor Biopsy @ Massachusetts General Hospital
With the advent of precision oncology, including both small molecule inhibitors targeting specific genetic drivers of cancer and modulators of the immune response to cancer neoantigens, there is a pressing need for advanced diagnostics to direct and monitor therapeutic interventions. Currently, such real time monitoring is performed through repeat, or resistance, biopsies; however, these surgical biopsies are invasive, may have significant complications including insufficient tissue for the intended analyses, and only sample one specific site of tumor, which may not be representative of the entire tumor cell population within a patient. Liquid biopsies are poised to revolutionize cancer therapies, by enabling frequent blood-based monitoring of tumor-derived materials, as cancers evolve in response to therapeutic interventions. The technological hurdle in isolating sufficient numbers of rare CTCs from routine blood specimens has been the single major limitation preventing the clinical deployment of CTC-based diagnostic opportunities. Enabled by the technology proposed here, our shift from processing routine 10 mL blood tubes (0.2% of whole blood volume) to making use of clinical leukapheresis to sample near-whole blood volumes (40-100%) addresses this challenge. The fundamental basis of the technology is the highly efficient depletion of antibody-tagged blood cells away from unmanipulated CTCs (?negative depletion? as opposed to ?positive selection?), thereby enabling tumor antigen? independent enrichment of unperturbed, viable CTCs. This strategy is applicable to cells disseminated from virtually any solid tumor type without making any a priori assumption on the physical or biological properties of tumor cells. Our hypothesis is that liquid biopsy of large number of CTCs using leukopheresis is equivalent to the invasive biopsies of metastatic tumor lesions, currently performed at the time of on-treatment disease progression in lung cancer. To address our hypothesis, we formulated 3 Aims. In Aim 1, we will develop a large-volume CTC isolation technology based on microfluidic negative selection. In Aim 2, we will integrate microfluidic components designed in Aim 1 into a monolithic chip for sorting of CTCs from leukopaks. In Aim 3, we will test our hypothesis by a direct comparison of tumor biopsy and liquid biopsy, performed within a few weeks of each other, assessing both their success rate and diagnostic accuracy. We believe that the convergence of resources and multidisciplinary expertise available in our team will lead to a transformative bioengineering technology, paired with a highly clinically-relevant clinical challenge, providing a new tool for preclinical lung cancer therapeutics. A positive outcome in this pilot study would set the stage for testing more complex clinical applications, ranging from measuring cancer cell signaling effects of drug therapy (?noninvasive pharmacokinetics?) and quantitation of cell surface protein targets for cancer immunotherapy (protein- based predictive markers), and ultimately even enable detection and tissue localization of early invasive cancers in at-risk individuals such as familial breast cancer, people with environmental risks (e.g., lung nodules in smokers, liver cancer in cirrhosis), abnormal radiographic findings of unknown significance or inaccessible biopsy.
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0.907 |
2021 |
Haber, Daniel A. Maheswaran, Shyamala Toner, Mehmet |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
High-Flow Microfluidics of Leukapheresis Blood Products For Functional Analysis of Breast Circulating Tumor Cells @ Massachusetts General Hospital
Liquid biopsies are poised to revolutionize cancer therapies, by enabling frequent blood-based monitoring of tumor-derived materials, as cancers evolve in response to therapeutic interventions. Nowhere is this more evident than in triple negative metastatic breast cancer, where women may undergo multiple serial courses of therapy over many years, each associated with an initial response, followed by the acquisition of drug resistance. Plasma DNA (ctDNA) mutation analyses provide a measure of tumor burden and may identify targetable mutations for small molecule inhibitors, but they do not allow ex vivo culture of intact circulating tumor cells (CTCs) for individualized preclinical drug sensitivity (?precision oncology?). Isolation of CTCs has been achieved using small blood volumes (5-10 mL), but these cells are so rare that it has not been feasible to establish clinically robust ex vivo CTC cultures. Our hypothesis is that we can massively increase the amount of blood screened through standard clinical leukapheresis (1-2 L), and in return, achieve 100+-fold increase in the number of isolated CTCs to establish routine ex vivo culture of CTCs. It is also important to note that ex vivo CTC cultures obtained by sampling 1-2 L blood will reflect the ?real time? molecular subtype, genetic composition, and evolving drug sensitivity profile of an individual patient's metastatic breast cancer, including multiple lesions that together contribute to the blood-borne CTC population and the patient's overall tumor burden. We will develop a high-throughput microfluidic technology that achieves highly efficient depletion of tagged blood cells away from unmanipulated single and clustered CTCs and provides ?tumor independent enrichment? performance, which is applicable to cells disseminated from any solid tumor type without making any a priori assumption on the physical and/or biological properties of tumor cells. We will also establish the microenvironmental conditions conducive to high efficiency ex vivo CTC cultures from women with metastatic triple negative breast cancer. Success would be transformative both for new drug development and for individualized patient selection among existing therapies for breast cancer patients, and others.
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0.907 |