It is generally believed that shutting down the kinase activity of BCR-ABL by imatinib will completely inhibit its functions, leading to inactivation of its downstream signaling pathways and cure of the disease. Imatinib is highly effective at treating human Philadelphia chromosome-positive (Ph ؉ ) chronic myeloid leukemia (CML) in chronic phase but not Ph ؉ B cell acute lymphoblastic leukemia (B-ALL) and CML blast crisis. We find that SRC kinases activated by BCR-ABL remain fully active in imatinib-treated mouse leukemic cells, suggesting that imatinib does not inactivate all BCR-ABLactivated signaling pathways. This SRC pathway is essential for leukemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Inhibition of both SRC and BCR-ABL kinase activities by dasatinib affords complete B-ALL remission. However, curing B-ALL and CML mice requires killing leukemic stem cells insensitive to both imatinib and dasatinib. Besides BCR-ABL and SRC kinases, stem cell pathways must be targeted for curative therapy of Ph ؉ leukemia.dasatinib ͉ imatinib ͉ SRC kinases

Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and Kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. Here we identified SHARPIN as an important inactivator of β1-integrins in an RNAi-screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity which was fully rescued by re-expression of SHARPIN. SHARPIN directly bound to a conserved cytoplasmic region of integrin α-subunits and inhibited recruitment of Talin and Kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.

T cells proteolytically shed the ectodomains of several cell surface proteins and, thereby, can alter their responsiveness and can release soluble intercellular regulators. ART2.2 is a GPI-anchored ecto-ADP-ribosyltransferase (ART) related to ADP-ribosylating bacterial toxins. ART2.2 is expressed exclusively by mature T cells. Here we show that ART2.2 is shed from the cell surface in enzymatically active form upon activation of T cells. Shedding of ART2.2 resembles that of L-selectin (CD62L) in dose response, kinetics of release, and sensitivity to the metalloprotease inhibitor Immunex Compound 3, suggesting that ART2.2, like CD62L, is cleaved by TNF-α-converting enzyme or by another metalloprotease. ART2.2 shed from activated T cells migrates slightly faster in SDS-PAGE analyses than does ART2.2 released upon cleavage of the GPI anchor. This indicates that shedding of ART2.2 is mediated by proteolytic cleavage close to its membrane anchor. Shed ART2.2 is enzymatically active and ADP-ribosylates several substrates in vitro. Thus, shedding of ART2.2 releases a potential intercellular regulator. Finally, using a new FACS assay for monitoring ADP-ribosylation of cell surface proteins, we demonstrate that shedding of ART2.2 correlates with a reduced sensitivity of T cell surface proteins to ADP-ribosylation. Our findings suggest that by shedding ART2.2 the activated T cell not only releases a potential intercellular regulator but also may alter its responsiveness to immune regulation by ART2.2-mediated ADP-ribosylation of cell surface proteins.

Background: Ischemic heart disease is a leading cause of heart failure and despite advanced therapeutic options, morbidity and mortality rates remain high. Although acute inflammation in response to myocardial cell death has been extensively studied, subsequent adaptive immune activity and anti-heart autoimmunity may also contribute to the development of HF. After ischemic injury to the myocardium, dendritic cells (DC) respond to cardiomyocyte necrosis, present cardiac antigen to T cells and potentially initiate a persistent autoimmune response against the heart. Cross-priming DC have the ability to activate both CD4+ helper and CD8 + cytotoxic T cells in response to necrotic cells and may thus be crucial players in exacerbating autoimmunity targeting the heart. This study investigates a role for cross-priming DC in post-MI myocardial impairment through presentation of self-antigen from necrotic cardiomyocytes to cytotoxic CD8 + T cells. Methods: We induced type-2 myocardial infarction (MI)-like ischemic injury in the heart by treatment with a single high dose of the beta-adrenergic agonist isoproterenol. We characterized the DC population in the heart and mediastinal lymph nodes and analyzed long-term cardiac immunopathology and functional decline in wild type and Clec9a -depleted mice lacking DC cross-priming function. Results: A diverse DC population, including cross-priming DC, is present in the heart and activated after ischemic injury. Clec9a -/- mice deficient in DC cross-priming are protected from long-term immune-mediated myocardial damage and decline of cardiac function, likely due to dampened activation of cytotoxic CD8 + T cells. Conclusions: Activation of cytotoxic CD8 + T cells by cross-priming DC contributes to exacerbation of post-ischemic inflammatory damage of the myocardium and corresponding decline in cardiac function. Importantly, this provides novel therapeutic targets to prevent immune-mediated worsening of post-ischemic HF.

Diminished stem-cell functions with age may be a major cause of anemias and other defects. Unfortunately, treatments that increase stem-cell function can also increase the incidence of cancers. Lifelong dietary restriction (DR) is known to decrease spontaneous cancers and lengthen lifespan. This study examines the effect of DR on the ability of bone marrow cells to repopulate irradiated recipients and produce erythrocytes and lymphocytes. In BALB/cByJ (BALB) mice, repopulating abilities decline with age; DR ameliorates this trend. In C57BL/6J (B6) and (BALB ؋ B6) F1 hybrid (F1) mice, repopulating abilities increase with age; DR maintains this increase. Hematopoietic stem cell (HSC) numbers are highly variable in aged BALB mice; however, the observed loss of marrow function results from a major loss in repopulating ability per HSC. DR greatly ameliorates this loss of function with age. In contrast, function per HSC in B6 mice is affected neither by age nor by DR. Thus, DR increases or maintains increased marrow repopulating ability with age in the 3 different genotypes tested, but effects on function per HSC depend on genotype. That DR increases or maintains stem-cell function with age, while decreasing cancer, has far-reaching health implications. IntroductionDecline in repopulating and differentiating abilities of hematopoietic stem cells (HSCs) is characteristic of human diseases such as aplastic anemia and bone marrow (BM) failure. The common unexplained anemias 1 in elderly people can be caused by declining HSC functions with age. These anemias are serious diseases with high financial and personal costs. 2,3 The functional defects found in HSCs may be an example of a more generalized intrinsic loss of proliferative capacity that occurs in all aged adult stem cells. 4,5 Thus, interventions that maintain HSC function with age will help alleviate these serious diseases and benefit health.Laboratory mice provide well-recognized models in which to study hematopoietic defects. Genetic regulation of the age-related decline in BM repopulating and differentiating abilities has been demonstrated by the in vivo comparison of these abilities among mouse strains. [6][7][8][9][10] The ability of bone marrow cells (BMCs) to repopulate and produce erythrocytes and lymphocytes is reduced in old DBA/2J (D2) and old BALB/cByJ (BALB) compared with young controls. However, BM from old C57BL/6J (B6) mice actually has a greater repopulating ability than BM from young controls, 6-10 although its ability to repopulate after serial transplantation diminishes. 6,11,12 These strain-related differences are apparently the result of the underlying genetic differences in stem-cell exhaustion. 11 Because the patterns of aging differ among mouse strains, it is important to examine HSC aging in more than a single strain of mice.The maximal potential lifespan of an organism may in part be determined by the increased risk of cancer concomitant with mechanisms that can maintain stem-cell function in aging subjects. 12,13 Thus, the loss of funct...