Jose A. Cancelas

Rac GTPases differentially integrate signals regulating hematopoietic stem cell localization

The molecular events that regulate engraftment and mobilization of hematopoietic stem cells and progenitors (HSC/Ps) are still incompletely defined. We have examined the role of the Rho GTPases Rac1 and Rac2 in HSC engraftment and mobilization. Rac1, but not the hematopoietic-specific Rac2, is required for the engraftment phase of hematopoietic reconstitution, because Rac1−/− HSCs did not rescue in vivo hematopoiesis after transplantation, but deletion of Rac1 after engraftment did not impair steady-state hematopoiesis. Rac1−/− HSC/Ps showed impaired spatial localization to the endosteum but near-normal homing to the medullary cavity in vivo. Interaction with the bone marrow microenvironment in vitro was markedly altered. Whereas post-engraftment deletion of Rac1 alone did not impair hematopoiesis, deficiency of both Rac1 and Rac2 led to massive mobilization of HSCs from the marrow associated with ineffective hematopoiesis and intense selection for Rac-expressing HSCs. This mobilization was reversible by re-expression of Rac1. In addition, a rationally designed, reversible small-molecule inhibitor of Rac activation led to transient mobilization of engraftable HSC/Ps. Rac proteins thus differentially regulate engraftment and mobilization phenotypes, suggesting that these biological processes and steady-state hematopoiesis are biochemically separable and that Rac proteins may be important molecular targets for stem cell modification.

Microenvironment Determines Lineage Fate in a Human Model of MLL-AF9 Leukemia

Faithful modeling of mixed-lineage leukemia in murine cells has been difficult to achieve. We show that expression of MLL-AF9 in human CD34+ cells induces acute myeloid, lymphoid, or mixed-lineage leukemia in immunodeficient mice. Some leukemia stem cells (LSC) were multipotent and could be lineage directed by altering either the growth factors or the recipient strain of mouse, highlighting the importance of microenvironmental cues. Other LSC were strictly lineage committed, demonstrating the heterogeneity of the stem cell compartment in MLL disease. Targeting the Rac signaling pathway by pharmacologic or genetic means resulted in rapid and specific apoptosis of MLL-AF9 cells, suggesting that the Rac signaling pathway may be a valid therapeutic target in MLL-rearranged AML.

Stem Cell Collection and Gene Transfer in Fanconi Anemia

Fanconi anemia (FA) is a rare genetic syndrome characterized by progressive bone marrow failure (BMF), congenital anomalies, and a predisposition to malignancy. Successful gene transfer into hematopoietic stem cells (HSCs) could reverse BMF in this disease. We developed clinical trials to determine whether a sufficient number of CD34+ stem cells could be collected for gene modification and to evaluate the safety and efficacy of HSC-corrective gene transfer in FA genotype A (FANCA) patients. Here, we report that FA patients have significant depletion of their BM CD34+ cell compartment even before severe pancytopenia is present. However, oncoretroviral-mediated ex vivo gene transfer was efficient in clinical scale in FA-A cells, leading to reversal of the cellular phenotype in a significant percentage of CD34+ cells. Re-infusion of gene-corrected products in two patients was safe and well tolerated and accompanied by transient improvements in hemoglobin and platelet counts. Gene correction was transient, likely owing to the low dose of gene-corrected cells infused. Our early experience shows that stem cell collection is well tolerated in FA patients and suggests that collection be considered as early as possible in patients who are potential candidates for future gene transfer trials.

Rho GTPase Cdc42 coordinates hematopoietic stem cell quiescence and niche interaction in the bone marrow

Adult hematopoietic stem cells (HSCs) exist in a relatively quiescent state in the bone marrow (BM) microenvironment to fulfill long-term self-renewal and multilineage differentiation functions, an event that is tightly regulated by extrinsic and intrinsic cues. However, the mechanism coordinating the quiescent state of HSCs and their retention in the BM microenvironment remains poorly understood. In a conditional-knockout mouse model, we show that Cdc42−/− HSCs enter the active cell cycle, resulting in significantly increased number and frequency of the stem/progenitor cells in the BM. Cdc42 deficiency also causes impaired adhesion, homing, lodging, and retention of HSCs, leading to massive egress of HSCs from BM to distal organs and peripheral blood and to an engraftment failure. These effects are intrinsic to the HSCs and are associated with deregulated c-Myc, p21Cip1, β1-integrin, and N-cadherin expressions and defective actin organization. Thus, Cdc42 is a critical coordinator of HSC quiescence maintenance and interaction with the BM niche.

Plexiform and Dermal Neurofibromas and Pigmentation Are Caused by Nf1 Loss in Desert Hedgehog-Expressing Cells

Neurofibromatosis type 1 (Nf1) mutation predisposes to benign peripheral nerve (glial) tumors called neurofibromas. The point(s) in development when Nf1 loss promotes neurofibroma formation are unknown. We show that inactivation of Nf1 in the glial lineage in vitro at embryonic day 12.5 + 1, but not earlier (neural crest) or later (mature Schwann cell), results in colony-forming cells capable of multilineage differentiation. In vivo, inactivation of Nf1 using a DhhCre driver beginning at E12.5 elicits plexiform neurofibromas, dermal neurofibromas, and pigmentation. Tumor Schwann cells uniquely show biallelic Nf1 inactivation. Peripheral nerve and tumors contain transiently proliferating Schwann cells that lose axonal contact, providing insight into early neurofibroma formation. We suggest that timing of Nf1 mutation is critical for neurofibroma formation.

Neuroblastoma Cell Lines Contain Pluripotent Tumor Initiating Cells That Are Susceptible to a Targeted Oncolytic Virus

Background Although disease remission can frequently be achieved for patients with neuroblastoma, relapse is common. The cancer stem cell theory suggests that rare tumorigenic cells, resistant to conventional therapy, are responsible for relapse. If true for neuroblastoma, improved cure rates may only be achieved via identification and therapeutic targeting of the neuroblastoma tumor initiating cell. Based on cues from normal stem cells, evidence for tumor populating progenitor cells has been found in a variety of cancers. Methodology/Principal Findings Four of eight human neuroblastoma cell lines formed tumorspheres in neural stem cell media, and all contained some cells that expressed neurogenic stem cell markers including CD133, ABCG2, and nestin. Three lines tested could be induced into multi-lineage differentiation. LA-N-5 spheres were further studied and showed a verapamil-sensitive side population, relative resistance to doxorubicin, and CD133+ cells showed increased sphere formation and tumorigenicity. Oncolytic viruses, engineered to be clinically safe by genetic mutation, are emerging as next generation anticancer therapeutics. Because oncolytic viruses circumvent typical drug-resistance mechanisms, they may represent an effective therapy for chemotherapy-resistant tumor initiating cells. A Nestin-targeted oncolytic herpes simplex virus efficiently replicated within and killed neuroblastoma tumor initiating cells preventing their ability to form tumors in athymic nude mice. Conclusions/Significance These results suggest that human neuroblastoma contains tumor initiating cells that may be effectively targeted by an oncolytic virus.

Rho GTPases in hematopoiesis and hemopathies

Rho family GTPases are intracellular signaling proteins regulating multiple pathways involved in cell actomyosin organization, adhesion, and proliferation. Our knowledge of their cellular functions comes mostly from previous biochemical studies that used mutant overexpression approaches in various clonal cell lines. Recent progress in understanding Rho GTPase functions in blood cell development and regulation by gene targeting of individual Rho GTPases in mice has allowed a genetic understanding of their physiologic roles in hematopoietic progenitors and mature lineages. In particular, mouse gene-targeting studies have provided convincing evidence that individual members of the Rho GTPase family are essential regulators of cell type-specific functions and stimuli-specific pathways in regulating hematopoietic stem cell interaction with bone marrow niche, erythropoiesis, and red blood cell actin dynamics, phagocyte migration and killing, and T- and B-cell maturation. In addition, deregulation of Rho GTPase family members has been associated with multiple human hematologic diseases such as neutrophil dysfunction, leukemia, and Fanconi anemia, raising the possibility that Rho GTPases and downstream signaling pathways are of therapeutic value. In this review we discuss recent genetic studies of Rho GTPases in hematopoiesis and several blood lineages and the implications of Rho GTPase signaling in hematologic malignancies, immune pathology. and anemia.

Structure-Function Based Design of Small Molecule Inhibitors Targeting Rho Family GTPases

Rho GTPases of the Ras superfamily are involved in the regulation of multiple cell functions and have been implicated in the pathology of various human diseases including cancer. They are attractive drug targets in future targeted therapy. A wealth of structure-function information made available by high resolution structures and mutagenesis studies has laid out the foundation for the derivation of a mechanism-based targeting strategy. Here we describe the rational design and characterizations of a first generation Rac-specific small molecule inhibitor. Based on the structure-function information of Rac interaction with GEFs, in a computer based Virtual Screening we have identified NSC23766, a highly soluble and membrane permeable compound, as a specific inhibitor of a subset of GEF binding to Rac and therefore Rac activation. In fibroblast cells NSC23766 inhibited Rac1 GTP-loading without affecting Cdc42 or RhoA activity and suppressed the Rac-GEF, Tiam1, and oncogenic Ras induced cell growth and transformation. NSC23766 also potently inhibited the prostate PC-3 cancer cell proliferation and invasion induced by Rac hyperactivation. Intraperitoneal administration of NSC23766 to laboratory mice resulted in effective Rac GTPase suppression and hematopoietic stem cell mobilization from the bone marrow to the peripheral blood, similar to the effects of genetically targeted disruption of Rac GTPases in the animals. A co-crystal structure of NSC23766 bound to Rac1 provided further insight for future medicinal chemistry modification and improvement of this lead Rac-specific inhibitor. Thus, structure-function based rational design may represent a new avenue for generating lead small molecule inhibitors of Ras superfamily GTPases that are useful for modulating pathological conditions in which the small GTPase deregulation may play a role.

Rac GTPase isoforms Rac1 and Rac2 play a redundant and crucial role in T-cell development

Rac GTPases have been implicated in the regulation of diverse functions in various blood cell lineages, but their role in T-cell development is not well understood. We have carried out conditional gene targeting to achieve hematopoietic stem cell (HSC)- or T-cell lineage-specific deletion of Rac1 or Rac1/Rac2 by crossbreeding the Mx-Cre or Lck-Cre transgenic mice with Rac1(loxp/loxp) or Rac1(loxp/loxp);Rac2(-/-) mice. We found that (1) HSC deletion of both Rac1 and Rac2 inhibited production of common lymphoid progenitors (CLPs) in bone marrow and suppressed T-cell development in thymus and peripheral organs, whereas deletion of Rac1 moderately affected CLP production and T-cell development. (2) T cell-specific deletion of Rac1 did not affect T-cell development, whereas deletion of both Rac1 and Rac2 reduced immature CD4(+)CD8(+) and mature CD4(+) populations in thymus as well as CD4(+) and CD8(+) populations in spleen. (3) The developmental defects of Rac1/Rac2 knockout T cells were associated with proliferation, survival, adhesion, and migration defects. (4) Rac1/Rac2 deletion suppressed T-cell receptor-mediated proliferation, IL-2 production, and Akt activation in thymocytes. Thus, Rac1 and Rac2 have unique roles in CLP production and share a redundant but essential role in later stages of T-cell development by regulating survival and proliferation signals.

Infectious complications in 126 patients treated with high-dose chemotherapy and autologous peripheral blood stem cell transplantation

The effect of an extensive prophylactic antimicrobial regimen was prospectively assessed in 126 patients after high-dose chemotherapy and autologous PBSC. They received ciprofloxacin (500 mg/12 h), acyclovir (200 mg/6 h), and itraconazole (200 mg/12 h) orally until neutrophil recovery. Febrile patients received i.v. imipenem (500 mg/6 h) to which vancomycin and amikacin were added if fever persisted for 2–3 and 5 days, respectively. Amphotericin B lipid complex was further given on day 7 or 8 of fever. Median times for a neutrophil count of >0.5 × 109/l and a platelet count of >20 × 109/l were 9 and 11 days. Severe neutropenia (<0.1 × 109/l) lasted for a median of 5 days in which 72% of febrile episodes and 50% of cases of bacteremia occurred. Gram- positive bacteria were isolated in 30 of 40 episodes of bacteremia, 25 of which were caused by Staphylococcus epidermidis. Clinical foci were the intravascular catheter in 35 cases, respiratory infection in 11, cellulitis in two, anal abscess in one, and neutropenic enterocolitis in one. The high incidence of febrile episodes (94%) and bacteremias (31%) may be due to the lack of efficacy of antimicrobial prophylaxis and the persistence of a 5-day period of severe neutropenia.