Human myeloproliferative diseases are common hematologic disorders characterized by clonal overproduction of maturing myeloid or erythroid cells, often caused by expression of a mutant, dysregulated tyrosine kinase (TK). These diseases can be accurately modeled in laboratory mice by the retroviral transfer of a mutant TK gene into murine hematopoietic stem and progenitor cells, followed by transplantation of these cells into irradiated recipient mice. This yields a model system for analyzing the molecular pathophysiology of these conditions and provides a platform for testing therapies, particularly molecularly targeted new chemical entities (NCEs). The Basic Protocol in this unit describes the preparation of mouse bone marrow cells to express the relevant human oncogene before transplanting them into irradiated recipient mice. An alternate protocol describes a similar technique that allows specific induction of lymphoproliferative disease by some TKs. Support protocols for generating and titering retroviral stocks are also included.

Among hematologic neoplasms, chronic myeloid leukemia (CML) is exquisitely sensitive to graft-versus-leukemia (GVL) because patients relapsing after allogeneic hematopoietic stem-cell transplantation (alloHSCT) can be cured by donor leukocyte infusion (DLI); however, the cellular mechanisms and strategies to separate GVL from GVHD are unclear. We used a BCR-ABL1 transduction/transplantation mouse model to study the mechanisms of DLI in MHC-matched, minor histocompatibility antigen-mismatched allogeneic chimeras with CML-like leukemia, in which DLI can be administered at the time of transplantation (early) or after recovery of hematopoiesis (delayed). After early DLI, CML-like leukemia cannot be transferred into immunocompetent secondary recipients as soon as 4 days after primary transplantation, demonstrating that cotransplantation of T lymphocytes blocks the engraftment of BCR-ABL1-transduced stem cells. In contrast, in allogeneic chimeras with established CML-like leukemia, combined treatment with delayed DLI and the kinase inhibitor imatinib eradicates leukemia with minimal GVHD. The GVL effect is directed against minor histocompatibility antigens shared by normal and leukemic stem cells, and is mediated predominantly by CD8+ T cells, with minor contributions from CD5- splenocytes, including natural killer cells. These results define a physiologic model of adoptive immunotherapy of CML that will be useful for investigating the cellular and molecular mechanisms of GVL.

Systemic inflammation arising from the organismal distribution of pathogen-associated molecular patterns is a major cause of clinical morbidity and mortality. Herein we report a critical and previously unrecognized in vivo role for germinal center kinase (GCK, genome nomenclature: map4k2), a mammalian Sterile 20 (STE20) orthologue, in PAMP signaling, and systemic inflammation. We find that disruption of gck in mice strongly impairs PAMP-stimulated macrophage cytokine and chemokine release and renders mice resistant to endotoxin-mediated lethality. Bone marrow transplantation studies show that hematopoietic cell GCK signaling is essential to systemic inflammation. Disruption of gck substantially reduces PAMP activation of macrophage Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs) via reduced activation of the MAPK-kinase-kinases (MAP3Ks) mixed lineage kinases (MLKs)-2 and -3. Extracellular signal-regulated kinase (ERK) and nuclear factor-kappaB (NF-kappaB) activation are largely unaffected. Thus, GCK is an essential PAMP effector coupling JNK and p38, but not ERK or NF-kappaB to systemic inflammation.

Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.