Combined inhibition of bile salt synthesis and intestinal uptake reduces cholestatic liver damage and colonic bile salts in mice.

Background & Aims: Intestine-restricted inhibitors of the apical sodium-dependent bile acid transporter (ASBT, or ileal bile acid transporter) are approved as treatment for several inheritable forms of cholestasis but are also associated with abdominal complaints and diarrhoea. Furthermore, blocking ASBT as a single therapeutic approach may be less effective in moderate to severe cholestasis. We hypothesised that interventions that lower hepatic bile salt synthesis in addition to intestinal bile salt uptake inhibition provide added therapeutic benefit in the treatment of cholestatic disorders. Here, we test combination therapies of intestinal ASBT inhibition together with obeticholic acid (OCA), cilofexor, and the non-tumorigenic fibroblast growth factor 15 (Fgf15)/fibroblast growth factor 19 (FGF19) analogue aldafermin in a mouse model of cholestasis. Methods: Wild-type male C57Bl6J/OlaHsd mice were fed a 0.05% 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet and received daily oral gavage with 10 mg/kg OCA, 30 mg/kg cilofexor, 10 mg/kg ASBT inhibitor (Linerixibat; ASBTi), or a combination. Alternatively, wild-type male C57Bl6J/OlaHsd mice were injected with adeno-associated virus vector serotype 8 (AAV8) to express aldafermin, to repress bile salt synthesis, or to control AAV8. During a 3-week 0.05% DDC diet, mice received daily oral gavage with 10 mg/kg ASBTi or placebo control. Results: Combination therapy of OCA, cilofexor, or aldafermin with ASBTi effectively reduced faecal bile salt excretion. Compared with ASBTi monotherapy, aldafermin + ASBTi further lowered plasma bile salt levels. Cilofexor + ASBTi and aldafermin + ASBTi treatment reduced plasma alanine transaminase and aspartate transaminase levels and fibrotic liver immunohistochemistry stainings. The reduction in inflammation and fibrogenesis in mice treated with cilofexor + ASBTi or aldafermin + ASBTi was confirmed by gene expression analysis. Conclusions: Combining pharmacological intestinal bile salt uptake inhibition with repression of bile salt synthesis may form an effective treatment strategy to reduce liver injury while dampening the ASBTi-induced colonic bile salt load. Impact and Implications: Combined treatment of intestinal ASBT inhibition with repression of bile salt synthesis by farnesoid X receptor agonism (using either obeticholic acid or cilofexor) or by expression of aldafermin ameliorates liver damage in cholestatic mice. In addition, compared with ASBT inhibitor monotherapy, combination treatments lower colonic bile salt load.

Complement-dependent cytotoxicity induced by therapeutic antibodies in B-cell acute lymphoblastic leukemia is dictated by target antigen expression levels and augmented by loss of membrane-bound complement inhibitors.

To optimally utilize therapeutic monoclonal antibodies in the treatment of B-cell acute lymphoblastic leukemia (B-ALL) understanding their mechanisms of action and the factors influencing these mechanisms is required. We show strong correlations between target antigen expression levels and sensitivity to complement-dependent cytotoxicity (CDC) induced by rituximab, ofatumumab, or alemtuzumab in a panel of cell lines derived from primary B-ALL cells and in primary B-ALL samples. Simultaneous loss of expression of membrane-bound complement regulatory proteins (mCRP) CD55 and CD59 due to glycophosphatidylinositol-anchor deficiency, significantly increased sensitivity to CDC. Accordingly, induced increase in CD55 or CD59 expression protected cells against CDC. The extent of protection co-depended on antigen expression and antibody concentration. In contrast, natural variation in mCRP expression could not be used as a single factor to predict sensitivity to CDC. In conclusion, sensitivity of B-ALL cells to CDC was predominantly determined by antibody concentration and target antigen expression.

Differential effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway in acute lymphoblastic leukemia.

PURPOSE: Aberrant PI3K/AKT/mTOR signaling has been linked to oncogenesis and therapy resistance in various malignancies including leukemias. In Philadelphia chromosome (Ph) positive leukemias, activation of PI3K by dysregulated BCR-ABL tyrosine kinase (TK) contributes to the pathogenesis and development of resistance to ABL-TK inhibitors (TKI). The PI3K pathway thus is an attractive therapeutic target in BCR-ABL positive leukemias, but its role in BCR-ABL negative ALL is conjectural. Moreover, the functional contribution of individual components of the PI3K pathway in ALL has not been established. EXPERIMENTAL DESIGN: We compared the activity of the ATP-competitive pan-PI3K inhibitor NVP-BKM120, the allosteric mTORC1 inhibitor RAD001, the ATP-competitive dual PI3K/mTORC1/C2 inhibitors NVP-BEZ235 and NVP-BGT226 and the combined mTORC1 and mTORC2 inhibitors Torin 1, PP242 and KU-0063794 using long-term cultures of ALL cells (ALL-LTC) from patients with B-precursor ALL that expressed the BCR-ABL or TEL-ABL oncoproteins or were BCR-ABL negative. RESULTS: Dual PI3K/mTOR inhibitors profoundly inhibited growth and survival of ALL cells irrespective of their genetic subtype and their responsiveness to ABL-TKI. Combined suppression of PI3K, mTORC1 and mTORC2 displayed greater antileukemic activity than selective inhibitors of PI3K, mTORC1 or mTORC1 and mTORC2. CONCLUSIONS: Inhibition of the PI3K/mTOR pathway is a promising therapeutic approach in patients with ALL. Greater antileukemic activity of dual PI3K/mTORC1/C2 inhibitors appears to be due to the redundant function of PI3K and mTOR. Clinical trials examining dual PI3K/mTORC1/C2 inhibitors in patients with B-precursor ALL are warranted, and should not be restricted to particular genetic subtypes.

Donor T cells administered over HLA class II barriers mediate antitumor immunity without broad off-target toxicity in a NOD/scid mouse model of acute leukemia.

Alloreactive (allo)-HLA-directed T cell responses after HLA-mismatched allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusion are typically considered detrimental responses mediating graft-versus-host disease (GVHD). Allo-HLA-reactive T cells with beneficial and selective graft-versus-leukemia (GVL) reactivity, however, can also be identified within an HLA-mismatched context. We investigated whether allo-HLA class II-directed T cells with beneficial GVL reactivity induced in NOD/scid mice engrafted with human chronic myelogenous leukemia in lymphoid blast crisis after treatment with donor lymphocyte infusion - mediated detrimental xenogeneic GVHD as a result of broad off-target cross-reactivity. The results demonstrate that beneficial GVL reactivity and xenogeneic GVHD are mediated by separate T cells. GVL reactivity was mediated by human T cells recognizing allo-HLA class II molecules, whereas xenoreactivity was exerted by human T cells recognizing H-2 molecules. Taken together, our data indicate a limited risk for detrimental off-target effects by allo-HLA class II-directed T cells and thereby provide a basis for the development of strategies for selecting allo-HLA-restricted T cells with selective GVL reactivity for adoptive transfer after HLA-mismatched allogeneic hematopoietic stem cell transplantation.

Imatinib mesylate at therapeutic doses has no impact on folliculogenesis or spermatogenesis in a leukaemic mouse model.

Imatinib should be avoided in women planning to become pregnant or during pregnancy, due to a higher risk of congenital malformations. However, it is not known whether imatinib affects future potential for fertility. Here we analysed ovaries and testes from adult mice receiving imatinib, focusing on testicular and ovarian functions. Seven male and 7 female mice were orally treated with 150 mg/kg body weight/day imatinib for two months. No effects on folliculogenesis or spermatogenesis could be observed postmortem by histological examinations, suggesting that, at least in two mouse models of imatinib treatment this tyrosine kinase inhibitor does not reduce fertility.

A mechanistic rationale for combining alemtuzumab and rituximab in the treatment of ALL.

B-lineage acute lymphoblastic leukemia (ALL) may express CD52 and CD20. Alemtuzumab (ALM) and rituximab (RTX) are therapeutic antibodies directed against CD52 and CD20, respectively, but showed limited activity against ALL in clinical trials. The mechanisms for the impaired responses remained unclear. We studied expression of CD52 and CD20 on ALL cells and found that most cases coexpressed CD52 and CD20. However, distinct CD52-negative (CD52(-)) subpopulations were detected in most cases as the result of defective glycophosphatidyl-inositol anchoring. Although ALM efficiently eradicated CD52-positive (CD52(+)) cells in NOD/scid mice engrafted with primary human ALL, CD52(-) subclones escaped therapy. In the same model, RTX showed limited activity resulting from occurrence of CD20 down-modulation. However, CD52(-) cells concurrently lacked the glycophosphatidyl-inositol-anchored complement regulators CD55 and CD59 and showed increased susceptibility to RTX-mediated complement-dependent cytotoxicity in vitro. At the same time, ALM was shown to inhibit down-modulation of CD20 in response to RTX by depleting the trogocytic capacity of phagocytic cells. Probably because of these complementary mechanisms, combined administration of ALM and RTX induced complete responses in vivo. Based on these data, we propose a mechanistic rationale for combined application of RTX and ALM in ALL.

Long-term culture of primary human lymphoblastic leukemia cells in the absence of serum or hematopoietic growth factors.

OBJECTIVE: B-lineage acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphatic blastic phase in adults have poor prognoses despite intensive chemotherapy. Novel targeted treatment modalities emerge, but their evaluation requires relevant in vitro models of lymphoblastic leukemia. Presently available cell lines do not fully represent this heterogeneous disease. Available in vitro culturing protocols do not support long-term proliferation of primary cells. We therefore aimed to develop a culture system that allows long-term proliferation of primary human B-lineage lymphoblastic leukemia. MATERIALS AND METHODS: Primary lymphoblastic leukemia cells were cultured in a defined serum-free medium, in the absence or presence of human hematopoietic growth factors or serum. RESULTS: In the defined serum-free medium, cells from 12 of 34 cases immediately proliferated in vitro. In the absence of hematopoietic growth factors and serum these cases proliferated for more than 1 year without signs of exhaustion. The culturing system supported different subtypes of lymphoblastic leukemia. Two chronic myeloid leukemia in lymphatic blastic phase, four bcr/abl-positive ALL, one etv6/abl-positive ALL, 2 e2a-pbx1-positive ALL, and one t(9;11)-positive ALL could be long-term expanded, as well as two ALL that displayed nontypical cytogenetics. Not all bcr/abl- or e2a-pbx1-positive ALL proliferated in vitro, demonstrating heterogeneity within these subtypes. The proliferating bcr/abl- and etv6/abl-positive cells displayed sensitivity to imatinib, demonstrating that their proliferation depended on the activity of these oncoproteins. CONCLUSION: The serum-free culturing system may be a valuable instrument in the study of ALL cell biology, as well as in the evaluation of novel targeted therapeutics.

Establishment and cytogenetic characterization of a human acute lymphoblastic leukemia cell line (ALL-VG) with ETV6/ABL1 rearrangement.

Fusion kinases (FK) like BCR/ABL1 mediate leukemic transformation and represent therapeutic targets. Fusion of ETV6 (ETS translocation variant 6, previously known as TEL) to ABL1 due to t(9;12) has been observed in various hematological malignancies. ETV6/ABL1 and BCR/ABL1 FK display similar activity but they may not be identical in function. Here we present the generation of an ETV6/ABL1 positive human acute lymphoblastic leukemia (ALL) cell line, ALL-VG. The cell line expressed ETV6/ABL1 fusion transcripts and displayed sensitivity to imatinib with an IC(50) of 0.1 microM. Karyotyping did not reveal overt t(9;12), suggesting a cryptic translocation. Fluorescent in situ hybridization and array-based comparative genomic hybridization were performed to characterize the rearrangement. ETV6/ABL1 fusion was demonstrated to result from insertion of a duplicated 300 to 1300 kb region of 9q34 that contained the distal portion of the ABL1 gene, into the ETV6 locus on 12p13. With this insertion, an 1150 to 1750 kb region of 12p13 that contained the distal portion of the ETV6 gene as well the cyclin dependent kinase inhibitor (CDKN) 1B gene was lost. Furthermore, the cells displayed a del(9)(p21.1 approximately p23), typically associated with loss of CDKN2A and CDKN2B. The ALL-VG cell line may serve as a tool for the study of ETV6/ABL1.

Limited antitumor-effect associated with toxicity of the experimental cytotoxic drug cyclopentenyl cytosine in NOD/scid mice with acute lymphoblastic leukemia.

The experimental cytotoxic drug cyclopentenyl cytosine (CPEC) is a non-competitive inhibitor of the enzyme cytidine triphosphate (CTP) synthethase. We evaluated the in vitro and in vivo antitumor activity of CPEC on human acute lymphoblastic leukemia (ALL) cell lines. CPEC displayed anti-leukemic activity with IC50 (after 3 days of incubation) ranging from 6 to 15 nM. Subsequently the in vivo activity of CPEC against primary human ALL was evaluated in a xenogeneic model of human ALL using NOD/scid mice inoculated with primary human ALL cells. In the model, only a marginal anti-leukemic activity was observed at 1.5 mg kg(-1) (5 days per week) and 5 mg kg(-1) (2 days per week), however, this activity was associated with severe systemic toxicity. The observed toxicity was not specific for the NOD/scid model, as toxicity at comparable treatment intensity was also observed in Balb/c mice. In conclusion, although CPEC showed antitumor activity against human ALL cells in vitro, its activity in the in vivo human leukemia model was only marginal and accompanied by severe toxicity.

Early detection and rapid isolation of leukemia-reactive donor T cells for adoptive transfer using the IFN-gamma secretion assay.

PURPOSE: The poor immunogenicity of most leukemias and the lack of specificity of the donor T cells limit the in vivo effectiveness of conventional donor lymphocyte infusions in many patients suffering from persistent or recurrent leukemia after allogeneic stem cell transplantation. These limitations may be overcome by the adoptive transfer of in vitro generated leukemia-reactive T cells. Although the potential clinical efficacy of this approach has been shown previously, lack of reproducibility of the procedure and the inability to show persistence and survival of the transferred T cells hampered further clinical application. The purpose of this study was to develop a new, broadly applicable strategy for the efficient generation and isolation of leukemia-reactive T cells with a better probability to survive and expand in vivo. EXPERIMENTAL DESIGN: Myeloid and B-cell leukemias were modified into professional immunogenic antigen-presenting cells, and used to stimulate HLA-matched donor T cells. After two stimulations, responding donor T cells were isolated based on their secretion of IFN-gamma and tested for their capacity to recognize and kill the primary leukemia. RESULTS: Using one universal stimulation and isolation protocol for various forms of leukemia, T-cell populations containing high frequencies of leukemia-reactive T cells could reproducibly be generated and early isolated under mild stimulatory conditions. Isolated T cells still had high proliferative potential and their reactivity seemed to be restricted to cells of the patient's hematopoiesis. CONCLUSION: We here show a new robust procedure for the generation and isolation of leukemia-reactive T cells for adoptive transfer.

Responses to donor lymphocyte infusion for acute lymphoblastic leukemia may be determined by both qualitative and quantitative limitations of antileukemic T-cell responses as observed in an animal model for human leukemia.

OBJECTIVE: Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation is largely unsuccessful in the treatment of acute lymphoblastic leukemia (ALL). To allow identification of the causes of this failure, we established an animal model of DLI in human ALL. METHODS: NOD/scid mice were inoculated with primary human ALL cells. Cells from five different patients were studied. After engraftment, DLI was performed by infusion of human leukocyte antigen (HLA)-identical donor T cells or HLA-disparate donor T cells. RESULTS: DLI resulted in expansion of activated, leukemia-reactive T cells in all donor-patient combinations. After 40 days of expansion, T cells abruptly declined in numbers and displayed loss of cytotoxicity. At this moment, remissions were observed in three of five donor-patient combinations. In animals engrafted with the two unresponsive ALL, remissions could be achieved when HLA-disparate DLI was given. CONCLUSION: Our results indicate that the inefficacy of DLI in ALL may be due to the limitation of the proliferative capacity of ALL-reactive T cells and that the antileukemic efficacy during the limited time span of proliferation depends on the antigenic disparity between the donor and the patient. The model can be used to study whether alternative strategies may result in more sustained antileukemic responses after DLI in ALL.

An animal model for human cellular immunotherapy: specific eradication of human acute lymphoblastic leukemia by cytotoxic T lymphocytes in NOD/scid mice.

Adoptive immunotherapy using in vitro-generated donor-derived cytotoxic T lymphocytes (CTLs) can be effective in the treatment of relapsed leukemia after allogeneic transplantation. To determine effector cell characteristics that result in optimal in vivo antileukemic efficacy, we developed an animal model for human CTL therapy. Nonobese diabetic/severe combined immunodeficiency (NOD/scid) mice were inoculated with either of 2 primary human acute lymphoblastic leukemia (ALL), denoted as SK and OF. Anti-SK and anti-OF CTLs were generated in vitro by repeated stimulation of donor peripheral blood mononuclear cells with either SK or OF cells. Both CTL lines displayed HLA-restricted reactivity against the original targets and non-major histocompatibility class (MHC)-restricted cross-reactivity in vitro. The CTLs were administered intravenously weekly for 3 consecutive weeks to mice engrafted with either SK or OF leukemia. In 3 of 8 SK-engrafted and anti-SK-treated mice, complete remissions were achieved in blood, spleen, and bone marrow. In the remaining 5 animals partial remissions were observed. In 4 of 4 OF-engrafted anti-OF-treated mice partial remissions were observed. The antileukemic effect of specific CTLs was exerted immediately after administration and correlated with the degree of HLA disparity of the donor-patient combination. In cross-combination-treated animals, no effect on leukemic progression was observed indicating that in vivo antileukemic reactivity is mediated by MHC-restricted effector cells. The CTLs, however, displayed an impaired in vivo proliferative capacity. Ex vivo analysis showed decreased reactivity as compared to the moment of infusion. We therefore conclude that the model can be used to explore the requirements for optimal in vivo efficacy of in vitro- generated CTLs.