Irradiated Normal Mice Research Articles

Targeting the Transcriptional Addiction of Leukemia Stem Cells By a New Class of Protein Kinase Inhibitors

Cancer stem cells present a major therapeutic challenge - their effective eradication depends on therapeutic targeting of the relative vulnerabilities of the cancer stem cells vs. normal tissue stem cells. One feature that distinguishes cancer initiating and propagating cells from their normal counterparts is transcriptional addiction, providing opportunities for novel therapeutic interventions with the aim of curing cancer.CKIα ablation appears as a promising means of activating p53 and killing leukemia cells in MDS and AML (Elyada et al , doi:10.1038/nature09673; Kronke et al, doi: 10.1038/nature14610); yet, with no selective CKIα inhibitors for in vivo use, the therapeutic value of CKIα inhibition cannot be validated. We succeeded in developing two different classes of CKIα inhibitors, one inducing CKIα degradation, and another targeting the catalytic pocket of CKIα, with no degradation. Both inhibitor types activate p53, yet we found that CKIα ablation or degradation was not sufficient to selectively eliminate leukemia stem cells, due to robust expression of anti-apoptotic oncogenes driven by super-enhancer activation.Through extensive medicinal chemistry, we obtained CKIα catalytic inhibitors with profound killing capacity of primary mouse AML cells and human AML cell lines. We demonstrated a strong therapeutic effect in a mouse model of MLL-AF9-induced leukemia, with >40% cure rates evident by transplantation of bone marrow from treated leukemic mice to lethally irradiated normal mice. All transplanted mice regained normal blood counts, exclusively derived from the donor bone marrow. None showed any evidence of residual disease for 6 months, demonstrating a successful therapeutic window, distinguishing leukemia stem cells (LSCs) from HSPCs. Parallel in vitro experiments, show that the inhibitors did not affect normal hematopoietic progenitors in a CFU assay, but eliminated AML progenitors at an IC50 <9nM.We found that CKIα inhibitors possessing potent anti-leukemia activity are distinguished by their capacity to co-target CDK9 and suppress the RNA Pol II elongation factor P-TEFb (CDK9-CyclinT1 complex), evident by blocking the phosphorylation of RNA Pol II C-terminal domain (pCTD2/5, see Figure). Co-crystallography studies elucidated the structural basis for co-targeting of CKIα and P-TEFb, the latter controls the rate limiting step of Poll-II-transcription and functions as a gatekeeper of super-enhancers. We found that both P-TEFb and super-enhancer activity are vigorously activated in AML cells. Therefore, the efficient P-TEFb-targeting activity of the inhibitors enables a selective blockade of leukemia super-enhancers (SE)-driven transcription, demonstrated by ChIP-seq analysis. As a result, SE-dependent transcription of major leukemia drivers including Myc and the anti-apoptotic oncogenes Bcl-2 and Mcl-1 was nearly abolished.Another remarkable feature of these dual CKIα-P-TEFb inhibitors is a rapid hit mechanism, by which, short drug exposure (10 mins in vitro and 2hrs in vivo) results in prolonged (24hrs) SE suppression and leukemia cell apoptosis. This unique property, which is at variance with the current occupancy-driven pharmacological paradigm, likely contributes to the dramatic therapeutic effect of co-targeting CKIα and P-TEFb in leukemia, whereby combination of rapid p53 induction with disruption of oncogenic super-enhancers eliminates leukemia stem cells without compromising normal HSPCs. The ability to disrupt super-enhancers and eliminate their associated oncogenes within minutes of drug exposure permits short intermittent therapy, which minimizes generalized toxicity. Indeed, at the therapeutic dose, we did not observe any significant adverse treatment effects even at daily treatment for 45 days in mice.To conclude, we developed a new class of small molecule inhibitors that co-targets CKIα and P-TEFb. These inhibitors have unique pharmacologic properties: short-term kinase inhibition results in long-term disruption of SE activity. Shutdown of leukemic super-enhancers in synergy with robust p53 activation compromises leukemic cells and stem cells addicted to SE-driven transcription. These features explain the powerful and specific anti-leukemic therapeutic effects of this new class of inhibitors in-vivo. [Display omitted] DisclosuresBen-Neriah:BioTheryX Inc.: Consultancy, Equity Ownership, Research Funding. Vacca:BioTheryX Inc.: Consultancy. Mercurio:BioTheryX: Employment, Equity Ownership.

Hematopoietic not systemic impairment of Roquin expression accounts for intestinal inflammation in Roquin-deficient mice

Roquin, an E3 ligase, is involved in curtailing autoimmune pathology as seen from studies using mice with mutated (Rc3h1san/san) or disrupted (Rc3h1gt/gt) Rc3h1 gene. The extent to which intestinal immunopathology is caused by insufficient Roquin expression in the immune system, or by Roquin impairment in non-hematopoietic cells, has not been determined. Using bone marrow cells from Rc3h1gt/gt mice transferred into irradiated normal mice (Rc3h1gt/gt → NL chimeras), we show that inflammation developed in the small intestine, kidney, lung, liver, and spleen. Proinflammatory cytokine levels were elevated in lamina propria lymphocytes (LPLs). Inflammation in the liver was accompanied by areas of hepatocyte apoptosis. Lung inflammation consisted of an influx of both T cells and B cells. Small intestinal LPLs had increased numbers of CD44hi, CD62Llo, KLRG1+, ICOS+ short-lived effector cells, indicating an influx of activated T cells. Following oral infection with L. monocytogenes, Rc3h1gt/gt → NL chimeras had more liver pathology and greater numbers of bacteria in the Peyer's patches than NL → NL chimeras. These findings demonstrate that small intestinal inflammation in Rc3h1san/san and Rc3h1gt/gt mice is due to a failure of Roquin expression in the immune system and not to insufficient systemic Roquin expression.

Generation and Characterization of Transgenic Mice Expressing MplW515L.

Abstract 3901Poster Board III-837Myeloproliferative neoplasias (MPNs) are a group of conditions characterized by chronic increases in some or all of the blood cells (platelets, white blood cells, and red blood cells). JAK2V617F, a gain-of-function mutation of tyrosine kinase JAK2, is found in over 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Attempt to identify other signaling components involving the JAK2 signaling pathways has led to discovery of acquired mutations of Mpl, the receptor of thrombopoietin, in 5-10% patients with PMF and ET. To prove the pathogenesis of Mpl mutants, we have generated transgenic mice expressing the most frequently occurred Mpl mutant designated MplW515L by using the vav gene promoter which drives expression of transgenes in the hematopoietic system. We obtained three lines of MplW515L transgenic mice which all displayed similar hematological abnormalities. As expected, the mice developed ET- and PMF-like phenotypes with much elevated platelet counts, severe splenomegaly/hepatomegaly, and bone marrow/spleen myelofibrosis. Interestingly, these mice also had markedly increased white blood cells in the peripheral blood, majority of which are IgD-positive mature B-cells. Histochemical staining and flow cytometric analyses revealed infiltrations of megkaryocytes and B cells into the spleen, the presence of megkaryocytes and erythroid blast cells in the liver, and infiltrations of the bone marrow with B-cells. Reticulin staining revealed that MplW515L transgenic mice developed profound myelofibrosis in the bone marrow and spleen. In vitro hematopoietic colony assays demonstrated increased numbers of hematopoietic progenitor cells including BFU-E, CFU-GM, CFU-Mk, and CFU-Pre-B in the bone marrow, mobilization of these stem/progenitor cells to peripheral blood and spleen, and their autonomous growth in the absence of growth factors and cytokines. Finally, transplantation of bone marrow cells from MplW515L mice into irradiated normal mice installed the aforementioned phenotypes into the recipient mice, indicating that expression of MplW515L altered the activity of hematopoietic stem cells. Together, our data demonstrated that transgenic expression of MplW515L not only causes PMF- and ET-like phenotypes but also lymphoproliferative disorders. Considering that Mpl is expressed in hematopoietic stem cells and that oncogenic gene mutations are often associated with alteration of gene expression, we believe that MplW515L may be involved in a wider spectrum of human hematological diseases than MPNs. Disclosures:No relevant conflicts of interest to declare.

Interference with nuclear factor kappa B and c-Jun NH2-terminal kinase signaling by TRAF6C small interfering RNA inhibits myeloma cell proliferation and enhances apoptosis

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of six adaptor proteins (TRAF1-6) links the TNFR superfamily to the nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1) transcriptional activators. Unlike other TRAFs, TRAF6 is also involved in Toll-like/interleukin (IL)-1 receptor (TIR) signal transduction. Thus, inhibition of TRAF6 function could interrupt both CD40 (TNFR family) and IL-1 growth signals, pathways critical to myeloma proliferation. To block TRAF6-mediated IL-1 signaling, we constructed small interfering RNA (siRNA) against TRAF6. We found that siRNA targeting the TRAF6 C-terminal (siTRAF6C) receptor interaction domain specifically reduced only TRAF6 protein expression, without affecting TRAF2 or 5 levels, and substantially interfered with IL-1-induced NF-kappaB and c-Jun/AP-1 activation. Inhibition by siTRAF6C was concentration-dependent. SiTRAF6C also significantly reduced myeloma proliferation and enhanced apoptosis in a similar dose-dependent fashion in vitro. More importantly, marked siTRAF6C growth inhibition was detected in vivo when these cells were implanted into the bone marrow of irradiated normal mice. In contrast, introduction of siRNA derived from the TRAF6 Zn-finger domain or an irrelevant siRNA construct failed to alter cell growth or cell death. These studies suggest that TRAF6 may be a new molecular target to block cell signal transduction important for the survival and proliferation of multiple myeloma cells.

Conversion of naive T cells to a memory-like phenotype in lymphopenic hosts is not related to a homeostatic mechanism that fills the peripheral naive T cell pool.

To examine directly whether a limited number of naive T cells transferred to lymphopenic hosts can truly fill the peripheral naive T cell pool, we compared the expansion and phenotype of naive T cells transferred to three different hosts, namely recombination-activating gene-deficient mice, CD3epsilon-deficient mice, and irradiated normal mice. In all three recipients, the absolute number of recovered cells was much smaller than in normal mice. In addition, transferred naive T cells acquired a memory-like phenotype that remained stable with time. Finally, injected cells were rapidly replaced by host thymic migrants in irradiated normal mice. Only continuous output of naive T cells by the thymus can generate a full compartment of truly naive T cells. Thus, conversion of naive T cells to a memory-like phenotype in lymphopenic hosts is not related to a homeostatic mechanism that fills the peripheral naive T cell pool.

IL-6 Undergoes Transition from in vitro Autocrine Growth Factor toin vivo Growth Inhibitor of B Lymphoma Cells.

IL-6 is a multifunctional cytokine involved in differentiation and proliferation of immune cells. Moreover, it has diverse effects on the proliferation of tumor cells in vivo and in vitro. Although stimulating cell growth of multiple myeloma cells, it inhibits the proliferation of B16 melanoma cells and lung cancer cells. B9.55 cells, B-cell lymphoma, are IL-6-dependent cells, definitely requiring exogenous IL-6 for growth. When the cDNA for IL-6 was transfected into B9.55 cells, they began growing in an autocrine pattern without exogenous IL-6. To investigate the effects of IL-6 on B9.55 lymphoma in vivo, IL-6-transfected B9.55 cells (B9.G7) or neotransfected B9.55 cells (B9.vec) were injected subcutaneously into syngeneic mice. Initially, B9.G7 outgrew B9.vec, but after 3 weeks, B9.G7 grew slower than B9.vec. In addition, 5 micro g of recombinant human IL-6 was injected daily into the tumor site. Reduced tumor sizes of IL-6-treated rats, similar to those observed in mice which received B9.G7, indicated that IL-6 itself is the mediator of tumor regression. When B9.G7 cells were injected into the irradiated normal mice, tumor regression was released compared with the untreated normal control, suggesting that radiosensitive host components were involved in the regression of B9.G7 cell growth. However, the tumor regression of B9.G7 cells was not released in SCID mice. Histologically, B9.G7 tumor demonstrated severe necrosis and apoptotic cells with infiltration of host inflammatory cells. Above data indicate that IL-6 functions as an autocrine growth factor for B9.G7 cells in vitro, but behaves as an autocrine inhibiting factor in vivo. These contrasting effects of IL-6 on tumor cells in vitro and in vivo will be facilitative in understanding the interaction of cytokines and host immune systems.

Hlx homeo box gene is essential for an inductive tissue interaction that drives expansion of embryonic liver and gut.

The divergent murine homeo box gene Hlx is expressed in restricted hematopoietic cell types and, during embryogenesis, prominently in visceral mesenchyme of the developing liver, gall bladder, and gut. Targeted disruption of the gene has now established that it plays a key role in visceral organogenesis. Embryos homozygous for the mutation died around embryonic day 15 with anemia and severe hypoplasia of the liver and gut. Liver ontogeny commenced normally with formation of the liver diverticulum and differentiation of hepatocytes, but the organ failed to expand and reached only 3% of normal size. The apparent liver hypoplasia was not associated with a notable increase in apoptotic cells. Gut development also began normally, but the intestines failed to undergo extensive elongation and looping and reached only a quarter of normal length. The anemia resulted from a deficiency in the fetal form of hematopoiesis, which occurs in the liver, but no intrinsic defect in Hlx-/- hematopoietic cells was observed in vitro, and liver-derived Hlx-/- hematopoietic stem cells that were transplanted to irradiated normal mice could fully reconstitute hematopoiesis. The impaired fetal hematopoiesis therefore reflects insufficient support function provided by the minute liver. Hlx is normally expressed in visceral mesenchyme lying adjacent to the developing liver and gut epithelia affected by the mutation, but not in the epithelia themselves. Hence, Hlx regulates a mesenchymal-epithelial interaction that drives a vital growth phase in visceral organogenesis. Moreover, because mutation of Hlx blocked liver growth but not its specification, early morphogenesis, or differentiation, development of this organ appears to occur by step-wise inductive interactions under separate genetic control.

Defect in negative selection in lpr donor-derived T cells differentiating in non-lpr host thymus.

Transplantation of bone marrow cells of lpr/lpr mice into irradiated normal mice fails to develop massive lymphadenopathy or autoimmunity but causes severe graft-vs.-host-like syndrome. To elucidate an abnormality of lpr/lpr bone marrow-derived T cells, we transplanted bone marrow cells of Mlsb lpr/lpr mice into H-2-compatible Mlsa non-lpr mice. Although lpr/lpr T cell precursors repopulated the host thymus as well as +/+ cells, a proportion of CD4+CD8+ cells decreased, and that of both CD4- and CD8- single-positive cells increased compared with those of +/+ recipients. Notably, in MRL/lpr----AKR and C3H/lpr----AKR chimeras, CD4 single-positive thymocytes contained an increased number of V beta 6+ cells in spite of potentially deleting alleles of Mlsa, whereas V beta 6+ mature T cells were deleted in the MRL/+ ----AKR and C3H/+ ----AKR chimeras. There was no difference between MRL/+ ----AKR and MRL/lpr----AKR chimeras in their proportion of V beta 3+ cells because both host and donor strain lack the deleting alleles. Interleukin 2 receptor expression of mature T cells, in the thymus and lymph node, was obviously higher in the MRL/lpr----AKR chimeras, in particular in the "forbidden" V beta 6+ subset. Moreover, lpr donor-derived peripheral T cells showed vigorous anti-CD3 response. These results indicate that lpr-derived T cells escape not only tolerance-related clonal deletion but also some induction of unresponsiveness in the non-lpr thymus.

Thymic involution and thymocyte phenotypic alterations induced by murine mammary adenocarcinomas.

A profound thymic atrophy has been observed in mice bearing large adenocarcinomas of the mammary gland. Only 2 to 5% of thymocytes remained 4 wk after tumor implantation. Although there is a slight decrease in the overall percentages of Thy-1+ cells in tumor bearers, the majority of the remaining cells are of a Thy-1 low phenotype. There was a lower percentage of double positive (CD4+, CD8+) cells, an increase of CD4+ CD8- thymocytes, similar percentages of CD4- CD8+ cells and double negative (CD4- CD8-) thymocytes in tumor-bearing mice. In addition, an increased percentage of CD3 cells could be detected in these animals. These results indicate that proportionally less immature thymocytes are present in the atrophic thymuses of mammary tumor bearers. Enhanced levels of glucocorticoids are known to produce similar effects on the thymus. However, adrenalectomy of mice followed by tumor implantation did not result in reversal of the thymic atrophy. Furthermore, a study of serum corticosterone levels in tumor bearers indicated no significant changes during tumorigenesis. A study of several parameters of bone marrow (BM) populations indicate that there is an increase in cells of the granulocyte-macrophage lineage and a decrease in lymphocytes induced by tumor-derived granulocyte macrophage-CSF. An alteration of prothymocytes in the BM is not the main cause of the thymic atrophy because BM cells from normal and tumor-bearing mice reconstituted irradiated normal mice equally well. There was no preferential recruitment of double positive cells to the spleen as indicated by no significant differences in the levels of T cells of immature phenotype including the CD4+ CD8+ population in the spleens of tumor bearers. Because no major changes were observed in tumor bearers, either at their capacity to repopulate the thymus or at the patterns of subsequent redistribution of thymocytes, it was postulated that the thymic atrophy may be caused by a direct or indirect effect of the tumor or tumor-associated factor(s). Intrathymic injections of tumor cells into young normal recipient mice resulted in a significant reduction of the thymus weight and cellularity. These data suggest that mammary tumors can secrete factor(s) that are capable of severely impairing the normal development of cells of the T cell lineage.

Improvements and limitations of the subrenal capsule assay for determining tumour sensitivity to cytostatic drugs

Cyclophosphamide (CY) prevented the host response from occurring in treated animals, and we therefore evaluated CY and other immunosuppressive forms of pretreatment in normal mice using the subrenal capsule assay initially for transplanted and later also for primary tumours. Cy pretreatment, 4 or 4.5 Gy whole-body irradiation and cortisone were superior to silica in reducing host cell infiltration, and irradiation as pretreatment has become our routine techique. The addition of cortisone acetate to irradiation was of minor benefit in only 1 3 transplanted lines. When primary tumours were tested, the irradiation was only rarely able to completely prevent cellular infiltration. Only 7 11 ovarian tumours and 3 9 lung tumours were evaluable as tumour specimens (>50% tumour) in preirradiated mice. The degree of infiltration and fibrosis was similar in transplants in irradiated normal mice or in athymic nude mice, suggesting that these phenomena are largely due to properties of the tumours rather than of the host. The limitations of the technique to some cell lines and occasional primary tumours is obvious.

Demonstration of bone marrow derived cells in synovial lining by means of giant intracellular granules as genetic markers.

Beige mice carry a gene (bg) which codes for the presence of giant intracellular granules in a variety of cell types. Bone marrow from beige mice was transplanted into irradiated normal mice. Giant granules similar to those seen in beige mouse synovial cells were observed subsequently in the synovial lining cells of marrow recipients, indicating an influx of bone marrow derived cells into the synovial lining. Giant granule bearing cells also appeared in the subintima. On electron microscopy giant granules have been demonstrated only in macrophage-like or type A cells in marrow recipient synovia, despite the occurrence of giant granules in both type A and type B lining cells in donor material (beige). This tends to suggest that only the type A lining cells are derived from bone marrow, as might be expected from their similarity to mononuclear phagocytes elsewhere. However, the possibility remains that type B cells are also derived from bone marrow but have a slower rate of replacement.

Marrow Transplantation and Iron Therapy in Mouse Hereditary Microcytic Anemia

AbstractThe defective tissues causing the microcytic anemia in mk/mk mice have been studied by transplantation of marrow erythrocyte precursors into lethally irradiated recipients. Microcytic (mk/mk) marrow in irradiated normal mice produced erythrocyte parameters, blood values, spleen/body weight ratios, and reticulocyte percentages characteristic of the microcytic anemia, but splenic iron stores remained high. Normal marrow in irradiated microcytic mice produced erythrocyte parameters intermediate between microcytic and normal and, combined with iron injections, produced normal erythrocyte parameters, blood values, spleen/body weight ratios, and reticulocyte percentages; long-term splenic iron stores remained low. Iron injections did not improve blood values of either irradiated normal mice implanted with microcytic marrow or untreated microcytic mice. Microcytic mice injected with iron recovered more rapidly from repeated severe bleedings than did uninjected microcytic mice, but iron injections made no difference in final blood values reached. Only slightly less 59Fe remained in mk/mk than in normal mice 22 days after injection. These results indicate that not only the erythrocyte precursor cells but also iron supplies to these cells and splenic iron stores are defective in microcytic mice and that iron loss is not responsible for the iron storage defect.

Radioprotective Effect of Rauscher Leukemia Virus in the SJL/J Mouse

Experiments have been performed to study the radiosensitivity of mice with Rauscher virus induced leukemia. Development of the leukemia was evaluated by measurements of the changes in spleen weight, leukocyte count and differentials, and hematocrit. Thirty-day survival of Rauscher virus injected mice was compared with that of normal mice following: no irradiation, 490, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 and 1100 R of x-rays. Thirty-day survival of Rauscher infected mice given no irradiation was only 36%. This was increased to 100% by exposure to 490 R of x-rays. From 490 to 750 R the effect of irradiation on both normal and Rauscher infected mice was equivalent. However, from 800 through 1050 R the leukemic mice showed a notably greater radioresistance than did irradiated normal mice. Thirty-day survival of leukemic mice after exposure to 800 R was twice as great as that of normal mice (80% vs. 40%). At 900 R and above none of the normal mice survived for 30 days after the irradiation. In contr...