Leukemia Xenografting Center

PI: Markus Müschen http://lymphoblasts.org/

Rationale: The Leukemia Xenografting Center is operated by the Müschen Laboratory at UCSF and funded by the Leukemia and Lymphoma Society (http://www.lls.org/#/) and the William Lawrence and Blanche Hughes Foundation (http://www.aspho.org/news/content/wlbhf.html/). The central goal of this initiative is to provide an interactive group of collaborators with a standardized platform to study human patient-derived leukemia cells.
Use this link for a full inventory of available leukemia samples (excel file).

The leukemia cells generated from this model system satisfy the following requirements:

1. Xenografted leukemia cells are representative of human disease. Commercially available cell lines have been grown in the presence of 21% oxygen and in the absence of bone marrow stroma for many years. The absence of the bone marrow microenvironment and the higher oxygen tension compared to bone marrow likely induces accumulation of ROS-mediated genetic lesions and outgrowth of subclones that differ from the original patient sample. Validation studies by us and others indicate that immediate engraftment of excess bone marrow samples from leukemia and myeloproliferative neoplasm patients into NOD/SCIDγc-/- mice results in phenotypically and genotypically stable human malignancies, which can be propagated over multiple passages.

2. Leukemia cells are available in abundance. The projects in this center require analyses of viable patient-derived leukemia cells for mechanistic studies. Since many of the mechanistic studies, including biochemical studies, involve large amounts of protein (e.g., immunoblot, co-immunoprecipitation), an expansion step of the patient-derived sample is necessary. Upon injection of 106 patient-derived leukemic bone marrow into sublethally irradiated NOD/γc-/- mice, we can harvest on average 200 x 106 leukemia cells from leukemic spleens of these mice. Since these cells maintain a stable genotype and phenotype across multiple passages, large numbers of cells can be generated from one patient sample.

3. Human leukemia cells can be studied in vivo in their physiologic environment. Work by us and others demonstrated that interactions between the bone marrow microenvironment and leukemia cells have a critical impact on the course of the disease and the risk of development of drug resistance. Ideally, development of leukemia should be studied in the context of a human bone marrow niche. A large body of evidence suggests that the mouse bone marrow environment represents a suitable model to study the development of human leukemia and responses to drug treatment in vivo.

4. Patient-derived leukemia xenografts represent a pre-clinical testing platform. Small molecule inhibitors developed in Core B can be readily tested in NOD/SCIDγc-/- mice that were engrafted with patient-derived leukemia cells. The results from these experiments will inform the development and design of clinical trials (LLS SCOR, PI Brian J Druker).

Description and Function of the Center: The goal of this Center is to generate xenografted leukemia samples for a group of interactive collaborators in sufficient numbers to aid in the mechanistic studies and to allow for a pre-clinical testing platform of kinase inhibitors. In collaboration with the Clinical Trials Core, we already have an IRB-approved Primary Patient Xenograft Repository. This multi-center collaboration has IRB approval at OHSU (Brian Druker), Children's Hospital Los Angeles and UCSF (Markus Müschen), and several other institutions. This core currently includes 40 bone marrow samples with full cytogenetic and immunophenotypic characterization. 25 of these samples have been successfully expanded (as of February 2010) through xenografting in immunocompromised NOD/SCIDγc-/- mice and are available to all SCOR investigators and investigators funded by the William Lawrence and Blanche Hughes Foundation (Table 1). This Center was initially established for ALL1,2. In the meantime, we have established xenografting protocols for AML3 which are based on work by John Dick's laboratory4 and others. In addition, we are in the process of establishing reproducible conditions for engraftment of MPNs. MPNs include chronic myeloid leukemia (CML), essential thrombocythemia (ET), polycythemia vera (PV) and primary (PMF) as most frequent subtypes. Among these MPNs, we achieved robust engraftment of human CML cells via intrafemoral injection2 (Figure 1). In addition, published data show feasibility of engraftment of hematopoeitic cells in PV and PMF5. Our preliminary data (Michael Deininger et al., unpublished) suggest that in PV and PMF engraftment efficiencies are variable. Therefore, a goal of Leukemia Xenografting Core is to establish optimal engraftment conditions for PV, PMF and other non-Ph MPN. We currently characterize patient-derived ALL samples via interphase FISH in our laboratory1 and have extended these analyses to AML and MPNs using a panel of FISH probes. In addition to cytogenetic and immunophenotypic characterization of the xenografted samples, gene expression data and SNP mapping array data (both Affymetrix) has been generated for each of the xenografted ALL and CML samples (Table 1). These data are linked to de-identified clinical information on treatment and outcome. All data is accessible to SCOR investigators and is hosted by the Bioinformatics Core at OHSU and UCSF (Dr. Huimin Geng) through a centralized data warehouse structure and file sharing servers.

Prioritization, choice of samples for xenografting and distribution among SCOR investigators. This core has the capability of generating approximately 30 primary leukemia samples per year. Samples selected for propagation in this core will be samples that have been identified from the siRNA and Kinase Inhibitor core (LLS SCOR, Brian J Druker) as candidates for mechanistic study in the various projects. Inclusion criteria will be neoplastic cells of >70% with 1 x 107 cells required for xenografting. Samples will be made available to SCOR investigators after verification of diagnosis and initial genotypic and phenotypic characterization of 2nd passage xenografts. Primary xenografts are not distributed to ensure that additional secondary xenografts can always be generated. Based on an injection of 1 million cells in a secondary recipient and a typical yield of 450 million leukemia cells from a leukemic spleen, we anticipate a 450-fold expansion for every secondary mouse recipient. We plan to engraft 3 secondary recipients simultaneously, which means a 1,350-fold expansion or an expected average yield of 1.35 x 109 leukemia cells from primary passage leukemia. Since leukemia samples can be efficiently expanded in secondary passage recipients, it is not necessary to implement a formal mechanism of prioritization for sample distribution among SCOR investigators: All samples will be made available as soon as validation of secondary xenografts has been completed. An updated directory of validated xenografts is available through a searchable web interface linked to the data warehouse structure (Bioinformatics; Dr. Huimin Geng).

Preclinical testing platform. Xenografting of leukemia cells from patient-derived bone marrow biopsies not only allows for amplification of sample size, it also represents a pre-clinical testing platform. Small molecule inhibitors identified in Core B can be readily tested in NOD/SCIDγc-/- mice that were engrafted with patient-derived leukemia cells. The results from these experiments will inform the development and design of clinical trials. As an example, we have established pre-clinical testing models for tyrosine kinase-driven leukemias (Ph+ ALL and CML)1,2 including in vivo experiments to test potential synergism of a retro-inverso BCL6 peptide inhibitor (RI-BPI)6 with tyrosine kinase inhibitors (TKI) against Ph+ ALL and CML. These experiments showed an almost complete clearance of leukemia burden after six injections (bioluminescence imaging) and significantly prolonged survival of RI-BPI-treated mice.

Table 1: Overview of xenografting of primary human ALL cells into NOD/SCIDγc-/- mice
Full inventory of available samples here (excel file)

Figure 1: Analysis of TEL-AML1 acute lymphoblastic leukemia

               A Bioluminescence                                                      B Immunohistochemistry

In this example, additional studies that can be performed include optimization of treatment schedules of the combination, determination of maximum tolerated dose levels, overall survival of mice, adverse effects and dose-limiting toxicities, along with detailed pharmacokinetics/pharmacodynamics. To make xenografted leukemia samples readily available to preclinical testing in this platform, for each validated leukemia sample, 107 cells will be transduced with lentiviral firefly luciferase, checked for luciferase activity and stored separately for this purpose.

Summary: The Primary Patient Xenografting Core is critical for the success of this Center's goals as it provides a centralized, IRB-approved repository of primary patient-derived leukemia samples . The availability of nearly unlimited access to patient-derived leukemia cells enables central translational aspects of all SCOR projects. As gene expression and biochemical (protein) analyses require large numbers of viable cells, having patient-derived cells that were expanded in NOD/SCIDγc-/- mice will be an essential resource for all projects. This Core also provides a critical pre-clinical testing platform. The goal of this Center is to identify new agents that interfere with oncogenic tyrosine kinase signaling i. and to tests these drugs in clinical trials. The pre-clinical testing platform within this core provides critical data that will inform prioritization of individual agents for further development and testing in a clinical setting.

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2. Klemm L, Duy C, Iacobucci I, Kuchen S, von Levetzow G, Feldhahn N, Henke N, Li Z, Hoffmann TK, Kim YM, Hofmann WK, Jumaa H, Groffen J, Heisterkamp N, Martinelli G, Lieber MR, Casellas R, Müschen M. The B cell mutator AID promotes B lymphoid blast crisis and drug resistance in chronic myeloid leukemia. Cancer Cell 2009 Sep 8; 16(3):232-45 View in: PubMed PDF Supplemental
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6. Cerchietti LC, Yang SN, Shaknovich R, Hatzi K, Polo JM, Chadburn A, Dowdy SF, Melnick A. A peptomimetic inhibitor of BCL6 with potent antilymphoma effects in vitro and in vivo. Blood. 2009; 113: 3397-405. View in: PubMed PDF