Tina M. Schnöder

Intracellular retention of ABL kinase inhibitors determines commitment to apoptosis in CML cells.

Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs.

Impact of FLT3-ITD location on sensitivity to TKI-therapy in vitro and in vivo.

OA Haabeth, A Tveita, M Fauskanger, K Hennig, PO Hofgaard and B Bogen Centre for Immune Regulation, Department of Immunology, Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Rikshospitalet, Oslo, Norway and KG Jebsen Centre for Influenza Vaccine Research, Department of Immunology, Institute of Clinical Medicine, University of Oslo and Oslo University Hospital, Rikshospitalet, Oslo, Norway E-mail: o.a.haabeth@medisin.uio.no These authors contributed equally to this work.

Clinically relevant doses of FLT3-kinase inhibitors quizartinib and midostaurin do not impair T-cell reactivity and function.

The vast majority of acute myeloid leukemia (AML) patients harboring an FLT3-ITD mutation experience relapse within a short period of time after discontinuation of chemotherapy.[1][1] Treatment options include experimental trials using FLT3-tyrosine kinase inhibitors (TKI) or allogeneic stem cell

Specificity of JAK-kinase inhibition determines impact on human and murine T-cell function

DOX (Supplementary Figure 7C). For in vivo therapy studies, mice bearing orthotopic MM tumors were treated with systemic administration of ApDCs or equal molar free DOX (Figure 2a). Changes in MM tumor size were monitored by whole-body bioluminescence imaging scans every 6 days (Figure 2b). Images at day 36 showed that ApDC treatment markedly inhibited orthotopic tumor growth, whereas free DOX had no effect under the same treatment conditions comparing to control group (Figure 2c). In addition, Kaplan–Meier survival analysis revealed that ApDCs treatment significantly improved survival rates of mice bearing MM tumors, indicating that ApDC was able to specifically deliver DOX to MM cells and accumulate high-DOX concentrations selectively within MM tumors for targeted therapy, although a sub-toxic concentration of DOX was administered to avoid off-target side effects. For histological examination, fresh cells from tumors were collected and cellular green fluorescent protein expression was detected by fluorescent microscopy (Figure 2e). Immunostaining of tumor tissues confirmed that MM tumor cells were CD38-positive and restricted for immunoglobulin lambda light chain expression (Figure 2f). Finally, analysis of healthy immunocompetent mice showed that ApDC treatment did not induce inflammation (Figure 2g), indicating suitability for in vivo therapy. Taken together, our study demonstrates that ApDCs are a new targeted therapeutic approach for specific delivery and intracellular release of a high drug payload under a unique pH-controlled mechanism in MM cells (Figure 1a). Our findings also indicate the clinical potential of ApDCs, which not only selectively inhibit MM tumors, but also have little or no off-target side effect in patients. To initiate clinical trials, further improvements and additional preclinical testing may be needed as discussed previously. Interestingly, this ApDC technology can be used as a universal platform to treat many cancers by simply replacing the aptamer sequence with one that targets different tumor biomarkers.

Activated protein C protects from GvHD via PAR2/PAR3 signalling in regulatory T-cells

Graft-vs.-host disease (GvHD) is a major complication of allogenic hematopoietic stem-cell(HSC) transplantation. GvHD is associated with loss of endothelial thrombomodulin, but the relevance of this for the adaptive immune response to transplanted HSCs remains unknown. Here we show that the protease-activated protein C (aPC), which is generated by thrombomodulin, ameliorates GvHD aPC restricts allogenic T-cell activation via the protease activated receptor (PAR)2/PAR3 heterodimer on regulatory T-cells (Tregs, CD4+FOXP3+). Preincubation of pan T-cells with aPC prior to transplantation increases the frequency of Tregs and protects from GvHD. Preincubation of human T-cells (HLA-DR4−CD4+) with aPC prior to transplantation into humanized (NSG-AB°DR4) mice ameliorates graft-vs.-host disease. The protective effect of aPC on GvHD does not compromise the graft vs. leukaemia effect in two independent tumor cell models. Ex vivo preincubation of T-cells with aPC, aPC-based therapies, or targeting PAR2/PAR3 on T-cells may provide a safe and effective approach to mitigate GvHD.Graft-vs.-host disease is a complication of allogenic hematopoietic stem cell transplantation, and is associated with endothelial dysfunction. Here the authors show that activated protein C signals via PAR2/PAR3 to expand Treg cells, mitigating the disease in mice.

Expression and function of ABC-transporter protein ABCB1 correlates with inhibitory capacity of Ruxolitinib in vitro and in vivo

The development of Janus kinase (JAK) inhibitors has revolutionized the therapeutic landscape for the treatment of myeloproliferative neoplasms (MPN). Therefore, improving the efficacy and safety of JAK inhibitor therapy is a major prerequisite for reliable clinical use in the future. Ruxolitinib (

JAK2-V617F promotes venous thrombosis through β1/β2 integrin activation

JAK2-V617F–positive chronic myeloproliferative neoplasia (CMN) commonly displays dysfunction of integrins and adhesion molecules expressed on platelets, erythrocytes, and leukocytes. However, the mechanism by which the 2 major leukocyte integrin chains, &bgr;1 and &bgr;2, may contribute to CMN pathophysiology remained unclear. &bgr;1 (&agr;4&bgr;1; VLA-4) and &bgr;2 (&agr;L&bgr;2; LFA-1) integrins are essential regulators for attachment of leukocytes to endothelial cells. We here showed enhanced adhesion of granulocytes from mice with JAK2-V617F knockin (JAK2+/VF mice) to vascular cell adhesion molecule 1– (VCAM1-) and intercellular adhesion molecule 1–coated (ICAM1-coated) surfaces. Soluble VCAM1 and ICAM1 ligand binding assays revealed increased affinity of &bgr;1 and &bgr;2 integrins for their respective ligands. For &bgr;1 integrins, this correlated with a structural change from the low- to the high-affinity conformation induced by JAK2-V617F. JAK2-V617F triggered constitutive activation of the integrin inside-out signaling molecule Rap1, resulting in translocation toward the cell membrane. Employing a venous thrombosis model, we demonstrated that neutralizing anti–VLA-4 and anti–&bgr;2 integrin antibodies suppress pathologic thrombosis as observed in JAK2+/VF mice. In addition, aberrant homing of JAK2+/VF leukocytes to the spleen was inhibited by neutralizing anti-&bgr;2 antibodies and by pharmacologic inhibition of Rap1. Thus, our findings identified cross-talk between JAK2-V617F and integrin activation promoting pathologic thrombosis and abnormal trafficking of leukocytes to the spleen.

Synergistic killing of FLT3ITD-positive AML cells by combined inhibition of tyrosine-kinase activity and N-glycosylation

Fms-like tyrosine kinase 3 (FLT3) with internal tandem duplications (ITD) is a major oncoprotein in acute myeloid leukemia (AML), and confers an unfavorable prognosis. Interference with FLT3ITD signaling is therefore pursued as a promising therapeutic strategy. In this study we show that abrogation of FLT3ITD glycoprotein maturation using low doses of the N-glycosylation inhibitor tunicamycin has anti-proliferative and pro-apoptotic effects on FLT3ITD-expressing human and murine cell lines. This effect is mediated in part by arresting FLT3ITD in an underglycosylated state and thereby attenuating FLT3ITD-driven AKT and ERK signaling. In addition, tunicamycin caused pronounced endoplasmatic reticulum stress and apoptosis through activation of protein kinase RNA-like endoplasmic reticulum kinase (PERK) and activation of the gene encoding CCAAT-enhancer-binding protein homologous protein (CHOP). PERK inhibition with a small molecule attenuated CHOP induction and partially rescued cells from apoptosis. Combination of tunicamycin with potent FLT3ITD kinase inhibitors caused synergistic cell killing, which was highly selective for cell lines and primary AML cells expressing FLT3ITD. Although tunicamycin is currently not a clinically applicable drug, we propose that mild inhibition of N-glycosylation may have therapeutic potential in combination with FLT3 kinase inhibitors for FLT3ITD-positive AML.

Diverging impact of cell fate determinants Scrib and Llgl1 on adhesion and migration of hematopoietic stem cells

PurposeCell fate determinants Scrib and Llgl1 influence self-renewal capacity of hematopoietic stem cells (HSCs). Scrib-deficient HSCs are functionally impaired and lack sufficient repopulation capacity during serial transplantation and stress. In contrast, loss of Llgl1 leads to increased HSC fitness, gain of self-renewal capacity and expansion of the stem cell pool. Here, we sought to assess for shared and unique molecular functions of Llgl1 and Scrib by analyzing their interactome in hematopoietic cells.MethodsInteractome analysis was performed by affinity purification followed by mass spectrometry. Motility, migration and adhesion were assessed on primary murine HSCs, which were isolated by FACS sorting following conditional deletion of Scrib or Llgl1, respectively. Imaging of Scrib-deficient HSCs was performed by intravital 2-photon microscopy.ResultsComparison of Scrib and Llgl1 interactome analyses revealed involvement in common and unique cellular functions. Migration and adhesion were among the cellular functions connected to Scrib but not to Llgl1. Functional validation of these findings confirmed alterations in cell adhesion and migration of Scrib-deficient HSCs in vitro and in vivo. In contrast, genetic inactivation of Llgl1 did not affect adhesion or migratory capacity of hematopoietic stem cells.ConclusionOur data provide first evidence for an evolutionarily conserved role of the cell fate determinant Scrib in HSC adhesion and migration in vitro and in vivo, a unique function that is not shared with its putative complex partner Llgl1.

Loss of DEP-1 (Ptprj) promotes myeloproliferative disease in FLT3-ITD acute myeloid leukemia

Acute myeloid leukemia (AML) is a heterogeneous group of diseases caused by oncogenic transformation of hematopoietic stem and progenitor cells. Mutations in the gene encoding the tyrosine kinase FLT3 leading to internal tandem duplications (ITDs) of sequence represent one of the most frequent