My research program focuses on the pathogenesis of lymphoid malignancy and entails two major arms. First, we have explored the role of NOTCH1 and other oncogenes/tumor suppressors in the genesis and propagation of T-cell acute lymphoblastic leukemia (T-ALL) including studies on downstream target genes/pathways and identifying mechanisms operative in leukemia stem cells. We have addressed these questions in cells from different developmental stages and tissue contexts on the hypothesis that preset epigenetic programs may restrict the oncogenic trajectories available to the cells as they undergo the initial stages of transformation and clonal establishment. Many of our findings have direct clinical relevance in that they serve as basis for the development of rational therapies that target disease-specific phenotypes.
As a second and more recent focus, my lab has explored the use of state-of-the-art mass cytometry (CyTOF) to obtain highly resolved phenotypic maps of heterogeneous cell populations in present in patient lymphoma biopsy samples including both malignant and reactive immune cell compartments. We have used this methodology to characterize intratumoral heterogeneity/subclonal diversity among malignant cell populations and stereotyped or patient-specific immune responses. This work is also of direct clinical relevance in providing detailed phenotypic characterizations that are required in order to define biomarkers for lymphoma classification and prognosis, and monitoring of patient-specific responses to therapy.
Specific projects include:
1) Pathogenetic mechanisms in T-ALL. Using our recently developed cord blood transduction approach, we are able to generate synthetic T-ALL by de novo transformation of normal human hematopoietic progenitors with activated NOTCH1 in combination with accessory oncogenes (M. Kusakabe et al, manuscript in preparation). This model produces clonal T-ALL populations in vitro within 2 months, or clonal T-ALL disease in mice within 3 months. Using this model, we are actively exploring the following areas:
- Epigenetic patterning in human T-ALL. We are characterizing the initial establishment and propagation of epigenetic patterns that direct the process of malignant transformation and clonal establishment. We are performing these studies in collaboration with Martin Hirst at UBC.
- Collaborating pathogenetic pathways in human T-ALL. We are executing shRNA and sgRNA screens to identify functional pathways required for clonal establishment and propagation.
- Cell-of-origin dependent programs T-ALL. By varying the input cell source, we are testing whether distinct types of T-ALL are generated from hematopoietic progenitors derived from cord blood vs. fetal liver vs. post-natal thymus vs. adult bone marrow. We have also done considerable work in this area using mouse tissues, comparing fetal liver and adult bone marrow, and revealed developmental stage-specific induction of autocrine growth factor signaling pathways (V. Giambra et al, manuscript in preparation).
- Clonal heterogeneity in human T-ALL. Using a cellular barcoding approach, we are exploring clonal dynamics during tumor establishment and propagation. We are performing this work in collaboration with Connie Eaves at BCCA.
*This work is currently funded by a Terry Fox Research Institute Program Project Grant (2012-2017).
2) Leukemia stem cells in T-ALL. We are continuing our work examining the roles of PKCθ and Wnt signaling in maintaining leukemia stem cell activity in this disease. In the PKCθ project, we have taken a biochemical approach to dissecting downstream pathways. We developed an analog-specific kinase variant of PKCθ, followed by discovery mode mass spectrometry (LC-ESI-MS/MS) to define direct kinase targets, and are pursuing functional validation of hits. This work is being done in collaboration with Gregg Morin at BCCA.
In the Wnt project, we are utilizing our integrated, real-time fluorescent Wnt reporter construct which labels a population of Wnt-active cells that is highly enriched for leukemia-initiating cell (LIC) activity, to define gene expression programs unique to leukemia stem cells. We are also dissecting the structural elements required for activating downstream gene expression programs using conditional Lef1 and Tcf7 mice obtained in collaboration with Howard Xue at the University of Iowa. We are also starting to extend this work into our synthetic human T-ALL model and in our sizable panel of patient-derived xenografts (PDX).
*This work is currently funded by a Canadian Institutes of Health Research (CIHR) Project Scheme operating grant (2016-2021).
3) Tumor ecosystem in human lymphoma. We are using mass cytometry (CyTOF) paired with single cell genomics approaches to characterize both inter- and intra-tumoral heterogeneity in human B-cell lymphomas. We are examining malignant B cell populations as well as infiltrating immune cell populations (predominantly T-cells). We are focusing this work on two common types of lymphoma, follicular lymphoma and diffuse large B-cell lymphoma. We access clinical specimens from the prodigious lymphoma tumor bank established at BCCA. We are performing these studies in collaboration with Sohrab Shah, Christian Steidl, and David Scott at BCCA.
*This project is currently funded by a Canadian Institutes of Health Research (CIHR) Project Scheme operating grant (2016-2021), and a Terry Fox Research Institute Program Project Grant (2016-2021).
Updated August 26, 2016
Currently Established Methodologies and Approaches
Animal models of leukemia/lymphoma including :
- Transgenic VavP-Bcl2 and IGF1Rneo (hypomorphic allele)
- Conditional (loxP/Cre) alleles for Cdkn2a, Prkcq, Runx1, Cbfb, Ctnnb1, Tcf7, and Lef1
- Viral transduction/bone marrow transplantation model
Xenograft animal models of leukemia including:
- Patient-derived xenograft models (16 lines established in the lab, access to an additional 10 by reciprocal agreement with other T-ALL labs)
- Synthetic human T-ALL generated by viral transduction/transplantation of human fetal liver, cord blood, post natal thymus, and adult bone marrow
Currently we use NSG as our standard immunodeficient recipients, but are currently testing other variants including human cytokine knock-ins/transgenics to support improved engraftment/propagation of human cells.
Cell culture models of leukemia/lymphoma including:
- Established human cell lines (~30 T-ALL and 10 DLBCL cell lines, all STR validated) and others including stromal feeders (OP9-DL1, OP9-DL4, MS5-DL1) and standard general use lines (293T, 3T3, U20S, HeLa, etc).
- PDX lines cultured on stromal feeders in defined serum-free media (Yost et al, Leukemia 2015).
- Primary cultures of virally-transduced human HSPC cultured on stromal feeders.
- In vitro adapted primary mouse leukemias.
Access to primary human lymphoma specimens:
I am Director of the BCCA Clinical Flow Cytometry Lab and co-manage the Lymphoma Tumor Repository with Drs. Christian Steidl and David Scott. The clinical flow lab accessions nearly 4,000 specimens each year for assessment of lymphoproliferative disease (LPD) including representative portions of ~1,000 excisional lymph node (LN) biopsies. Among these, approximately 100 per year represent follicular lymphoma (FL) and another 100 per year represent diffuse large B cell lymphoma (DLBCL). Single cell suspensions are generated by manual disaggregation and processed for flow cytometric phenotyping using our routine 13-color clinical assay (BD LSRFortessa platform). After diagnostic testing is completed, there are often several to tens of millions of excess viable cells remaining which are prospectively banked with DMSO cryoprotectant. This has been going on for over 2 decades as part of the well established BCCA Lymphoma Program. There are currently over 1,300 cases each of FL and DLBCL in the tumor bank, as well as abundant reactive (normal) lymph nodes to serve as a source of normal control material.
Gene transduction/editing approaches:
- Pseudotyped lentiviral vectors including use of multicistronic picornaviral 2A cassettes and fluorescent/epitope tags including GFP, YFP, mCherry, tNGFR, mCD8, hCD4, and hCD8
- Lentiviral shRNA library screening
- CRISPR/Cas9 ribonucleoprotein-mediated gene editing
- High parameter conventional flow cytometry, mass cytometry (CyTOF)
- qRT-PCR, droplet digital PCR (Bio-Rad QX200)
- WesTM western blotting
- Ascorbate peroxidase (APEX)-generated molecular labeling
- Analog-specific kinases (à la Kevan Shokat, in collaboration with Gregg Morin)
- Whole exome sequencing, targeted amplicon sequencing (in collaboration with Christian Steidl at BCCA)
- RNA-seq (in collaboration with Martin Hirst at UBC)
- ChIP-seq (in collaboration with Martin Hirst at UBC)
- Nano-electrospray ionization tandem mass spectrometry (LC-MS/MS) (in collaboration with Gregg Morin at BCCA)
Updated Jan 13, 2016