Kinase-inactivated CDK6 preserves the long-term functionality of adult hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are characterized by the ability to self-renew and to replenish the hematopoietic system. The cell-cycle kinase cyclin dependent-kinase 6 (CDK6) regulates transcription, whereby it has both kinase-dependent and kinase-independent functions. We here describe the complex role of CDK6, balancing quiescence, proliferation, self-renewal and differentiation in activated HSCs. Mouse HSCs expressing kinase-inactivated CDK6 show enhanced long-term repopulation and homing, whereas HSCs lacking CDK6 have impaired functionality. The transcriptomes of basal and serially transplanted HSCs expressing kinase-inactivated CDK6 exhibit an expression pattern dominated by HSC quiescence and self-renewal, proposing a concept where MAZ and NFY-A are critical CDK6 interactors. Pharmacologic kinase inhibition with a clinically used CDK4/6 inhibitor in murine and human HSCs validated our findings and resulted in increased repopulation capability and enhanced stemness. Our findings highlight a kinase-independent role of CDK6 in long-term HSC functionality. CDK6 kinase inhibition represents a possible strategy to improve HSC fitness.

Assessing the Accuracy of Sales Forecasts Submitted by Pharmaceutical Companies Applying for Reimbursement in Austria.

Objectives: Reimbursement decisions on new medicines require an assessment of their value. In Austria, when applying for reimbursement of new medicines, pharmaceutical companies are also obliged to submit forecasts of future sales. We systematically examined the accuracy of these pharmaceutical sales forecasts and hence the usefulness of these forecasts for reimbursement evaluations. Methods: We retrospectively analyzed reimbursement applications of 102 new drugs submitted between 2005 and 2014, which were accepted for reimbursement outside of hospitals, and for which actual reimbursed sales were available for at least 3 years. The main outcome variable was the accuracy ratio, defined as the ratio of forecasted sales submitted by pharmaceutical companies when applying for reimbursement to actual sales from reimbursement data. Results: The median accuracy ratio [95% confidence interval] was 1.33 [1.03; 1.74, range 0.15-37.5], corresponding to a median overestimation of actual sales by 33%. Forecasts of actual sales for 55.9% of all examined products either overestimated actual sales by more than 100% or underestimated them by more than 50%. The accuracy of sales forecasts did not show systematic change over the analyzed decade nor was it discernibly influenced by reimbursement status (restricted or unrestricted), the degree of therapeutic benefit, or the therapeutic area of the pharmaceutical product. Sales forecasts of drugs with a higher degree of innovation and those within a dynamic market tended to be slightly more accurate. Conclusions: The majority of sales forecasts provided by applicants for reimbursement evaluations in Austria were highly inaccurate and were on average too optimistic. This is in line with published results for other jurisdictions and highlights the need for caution when using such forecasts for reimbursement procedures.

Regimen-dependent synergism and antagonism of treprostinil and vildagliptin in hematopoietic cell transplantation.

The cell dose in umbilical cord blood units is a major determinant for the outcome of hematopoietic cell transplantation. Prostaglandin analogs and dipeptidylpeptidase-4 (DPP4/CD26)-inhibitors enhance the ability of hematopoietic stem cells (HSCs) to reconstitute hematopoiesis. Here we explored the synergism between treprostinil, a stable prostaglandin agonist, and the DPP4/CD26-inhibitor vildagliptin. The combination of treprostinil and forskolin caused a modest but statistically significant increase in the surface levels of DPP4/CD26 on hematopoietic stem and progenitor cells (HSPCs) derived from murine bone and human cord blood. Their migration towards stromal cell-derived factor-1 (SDF-1/CXCL12) was enhanced, if they were pretreated with treprostinil and forskolin, and further augmented by vildagliptin. Administration of vildagliptin rescued 25% of lethally irradiated recipient mice injected with a limiting number of untreated HSPCs, but 90 to 100% of recipients injected with HSPCs preincubated with treprostinil and forskolin. The efficacy of vildagliptin surpassed that of treprostinil (60% rescue). Surprisingly, concomitant administration of vildagliptin and treprostinil resulted in poor survival of recipients indicating mutual antagonism, which was recapitulated when homing of and colony formation by HSPCs were assessed. These observations of regimen-dependent synergism and antagonism of treprostinil and vildagliptin are of translational relevance for the design of clinical trials. KEY MESSAGES: Pretreatment with treprostinil increases surface levels of DPP4/CD26 in HSPCs. Vildagliptin enhances in vitro migration of pretreated HSPCs. Vildagliptin enhances in vivo homing and engraftment of pretreated HSPCs. Unexpected mutual antagonism in vivo by concomitant administration of vildagliptin and treprostinil.

Overexpression of PDE4A Acts as Checkpoint Inhibitor Against cAMP-Mediated Immunosuppression in vitro.

Malignant cells acquire physiological mechanisms of immunosuppression to escape immune surveillance. Strategies to counteract this suppression could help to improve adoptive immunotherapy regimen. The intracellular second messenger cyclic AMP (cAMP) acts as a potent immunosuppressive signaling molecule in T-cells and is up-regulated by multiple tumor-relevant suppressive factors including prostaglandin E2 (PGE2), adenosine and the functions of regulatory T-cells. Consequently, we aimed to abrogate cAMP signaling in human T-cells by ectopic overexpression of phosphodiesterase 4A (PDE4A). We could show that retroviral transduction of PDE4A into T-cells led to efficient degradation of cAMP in response to induction of adenylate cyclase. Retroviral transduction of PDE4A into CD4+ and CD8+ T-cells restored proliferation, cytokine secretion as well as cytotoxicity under immunosuppression by PGE2 and A2A-R agonists. PDE4A-transgenic T-cells were also partially protected from suppression by regulatory T-cells. Furthermore, PGE2-mediated upregulation of the inhibitory surface markers CD73 and CD94 on CD8+ T-cells was efficiently counteracted by PDE4A. Importantly, no differences in the functionality under non-suppressive conditions between PDE4A- and control-vector transduced T-cells were observed, indicating that PDE4A does not interfere with T-cell activation per se. Similarly, expression of surface markers associated with T-cell exhaustion were not influenced by PDE4A overexpression in long term cultures. Thus, we provide first in vitro evidence that PDE4A can be exploited as immune checkpoint inhibitor against multiple suppressive factors.

Treprostinil reduces endothelial damage in murine sinusoidal obstruction syndrome.

Sinusoidal obstruction syndrome (SOS) is a major complication after hematopoietic stem cell transplantation and belongs to a group of diseases increasingly identified as transplant-related systemic endothelial disease. Administration of defibrotide affords some protection against SOS, but the effect is modest. Hence, there is unmet medical need justifying the preclinical search for alternative approaches. Prostaglandins exert protective actions on endothelial cells of various vascular beds. Here, we explored the therapeutic potential of the prostacyclin analog treprostinil to prevent SOS. Treprostinil acts via stimulation of IP, EP2, and EP4 receptors, which we detected in murine liver sinusoidal endothelial cells (LSECs). Busulfan-induced cell death was reduced when pretreated with treprostinil in vitro. In a murine in vivo model of SOS, concomitantly administered treprostinil caused lower liver weight-to-body weight ratios indicating liver protection. Histopathological changes were scored to assess damage to liver sinusoidal endothelial cells, to hepatocytes, and to the incipient fibrotic reaction. Treprostinil indeed reduced sinusoidal endothelial cell injury, but this did not translate into reduced liver cell necrosis or fibrosis. In summary, our observations provide evidence for a beneficial effect of treprostinil on damage to LSECs but unexpectedly treprostinil was revealed as a double-edged sword in SOS. KEY MESSAGES: Murine liver sinusoidal endothelial cells (LSECs) express prostanoid receptors. Treprostinil reduces busulfan-induced cell death in vitro. Treprostinil lowers liver weight-to-body weight ratios in mice. Treprostinil positively affects LSECs in mice but not hepatic necrosis/fibrosis.

The impact of parallel regulatory-health technology assessment scientific advice on clinical development. Assessing the uptake of regulatory and health technology assessment recommendations.

AIMS: The parallel regulatory-health technology assessment scientific advice (PSA) procedure allows manufacturers to receive simultaneous feedback from both EU regulators and health technology assessment (HTA) bodies on development plans for new medicines. The primary objective of the present study is to investigate whether PSA is integrated in the clinical development programmes for which advice was sought. METHODS: Contents of PSA provided by regulators and HTA bodies for each procedure between 2010 and 2015 were analysed. The development of all clinical studies for which PSA had been sought was tracked using three different databases. The rate of uptake of the advice provided by regulators and HTA bodies was assessed on two key variables: comparator/s and primary endpoint. RESULTS: In terms of uptake of comparator recommendations at the time of PSA in the actual development, our analysis showed that manufacturers implemented comparators to address both the needs of regulators and of at least one HTA body in 12 of 21 studies. For primary endpoints, in all included studies manufacturers addressed both the needs of the regulators and at least one HTA body. CONCLUSIONS: One of the key findings of this analysis is that manufacturers tend to implement changes to the development programme based on both regulatory and HTA advice with regards to the choice of primary endpoint and comparator. It also confirms the challenging choice of the study comparator, for which manufacturers seem to be more inclined to satisfy the regulatory advice. Continuous research efforts in this area are of paramount importance from a public health perspective.

Adaptive Pathways: Possible Next Steps for Payers in Preparation for Their Potential Implementation.

Medicines receiving a conditional marketing authorization through Medicines Adaptive Pathways to Patients (MAPPs) will be a challenge for payers. The "introduction" of MAPPs is already seen by the European Medicines Agency (EMA) as a fait accompli, with payers not consulted or involved. However, once medicines are approved through MAPPs, they will be evaluated for funding by payers through different activities. These include Health Technology Assessment (HTA) with often immature clinical data and high uncertainty, financial considerations, and negotiations through different types of agreements, which can require monitoring post launch. Payers have experience with new medicines approved through conditional approval, and the fact that MAPPs present additional challenges is a concern from their perspective. There may be some activities where payers can collaborate. The final decisions on whether to reimburse a new medicine via MAPPs will have more variation than for medicines licensed via conventional processes. This is due not only to increasing uncertainty associated with medicines authorized through MAPPs but also differences in legal frameworks between member states. Moreover, if the financial and side-effect burden from the period of conditional approval until granting full marketing authorization is shifted to the post-authorization phase, payers may have to bear such burdens. Collection of robust data during routine clinical use is challenging along with high prices for new medicines during data collection. This paper presents the concept of MAPPs and possible challenges. Concerns and potential ways forward are discussed and a number of recommendations are presented from the perspective of payers.

Modulation of the heart's electrical properties by the anticonvulsant drug retigabine.

Retigabine, currently used as antiepileptic drug, has a wide range of potential medical uses. Administration of the drug in patients can lead to QT interval prolongation in the electrocardiogram and to cardiac arrhythmias in rare cases. This suggests that the drug may perturb the electrical properties of the heart, and the underlying mechanisms were investigated here. Effects of retigabine on currents through human cardiac ion channels, heterologously expressed in tsA-201 cells, were studied in whole-cell patch-clamp experiments. In addition, the drug's impact on the cardiac action potential was tested. This was done using ventricular cardiomyocytes isolated from Langendorff-perfused guinea pig hearts and cardiomyocytes derived from human induced pluripotent stem cells. Further, to unravel potential indirect effects of retigabine on the heart which might involve the autonomic nervous system, membrane potential and noradrenaline release from sympathetic ganglionic neurons were measured in the absence and presence of the drug. Retigabine significantly inhibited currents through hKv11.1 potassium, hNav1.5 sodium, as well as hCav1.2 calcium channels, but only in supra-therapeutic concentrations. In a similar concentration range, the drug shortened the action potential in both guinea pig and human cardiomyocytes. Therapeutic concentrations of retigabine, on the other hand, were sufficient to inhibit the activity of sympathetic ganglionic neurons. We conclude that retigabine- induced QT interval prolongation, and the reported cases of cardiac arrhythmias after application of the drug in a typical daily dose range, cannot be explained by a direct modulatory effect on cardiac ion channels. They are rather mediated by indirect actions at the level of the autonomic nervous system.

Hepatic Deletion of Janus Kinase 2 Counteracts Oxidative Stress in Mice.

Genetic deletion of the tyrosine kinase JAK2 or the downstream transcription factor STAT5 in liver impairs growth hormone (GH) signalling and thereby promotes fatty liver disease. Hepatic STAT5 deficiency accelerates liver tumourigenesis in presence of high GH levels. To determine whether the upstream kinase JAK2 exerts similar functions, we crossed mice harbouring a hepatocyte-specific deletion of JAK2 (JAK2Δhep) to GH transgenic mice (GHtg) and compared them to GHtgSTAT5Δhep mice. Similar to GHtgSTAT5Δhep mice, JAK2 deficiency resulted in severe steatosis in the GHtg background. However, in contrast to STAT5 deficiency, loss of JAK2 significantly delayed liver tumourigenesis. This was attributed to: (i) activation of STAT3 in STAT5-deficient mice, which was prevented by JAK2 deficiency and (ii) increased detoxification capacity of JAK2-deficient livers, which diminished oxidative damage as compared to GHtgSTAT5Δhep mice, despite equally severe steatosis and reactive oxygen species (ROS) production. The reduced oxidative damage in JAK2-deficient livers was linked to increased expression and activity of glutathione S-transferases (GSTs). Consistent with genetic deletion of Jak2, pharmacological inhibition and siRNA-mediated knockdown of Jak2 led to significant upregulation of Gst isoforms and to reduced hepatic oxidative DNA damage. Therefore, blocking JAK2 function increases detoxifying GSTs in hepatocytes and protects against oxidative liver damage.

Novel non-canonical role of STAT1 in Natural Killer cell cytotoxicity.

STAT1 is an important regulator of NK cell maturation and cytotoxicity. Although the consequences of Stat1-deficiency have been described in detail the underlying molecular functions of STAT1 in NK cells are only partially understood. Here, we describe a novel non-canonical role of STAT1 that was unmasked in NK cells expressing a Stat1-Y701F mutant. This mutation prevents JAK-dependent phosphorylation, subsequent nuclear translocation and cytokine-induced transcriptional activity as verified by RNA-seq analysis. As expected Stat1-Y701F mice displayed impaired NK cell maturation comparable to Stat1-/- animals. In contrast Stat1-Y701F NK cells exerted a significantly enhanced cytotoxicity in vitro and in vivo compared to Stat1-/- NK cells in the absence of detectable transcriptional activity. We thus investigated the STAT1 interactome using primary NK cells derived from Stat1ind mice that inducibly express a FLAG-tagged STAT1. Mass spectrometry revealed that STAT1 directly binds proteins involved in cell junction formation and proteins associated to membrane or membrane-bound vesicles. In line, immunofluorescence studies uncovered the recruitment of STAT1 to the target-cell interphase during NK cell killing. This led us to propose a novel function for STAT1 at the immunological synapse in NK cells regulating tumor surveillance and cytotoxicity.

Repurposing Treprostinil for Enhancing Hematopoietic Progenitor Cell Transplantation.

Activation of Gs-coupled receptors enhances engraftment of hematopoietic stem and progenitor cells (HSPCs). We tested the hypothesis that treprostinil, a prostacyclin analog approved for the treatment of pulmonary hypertension, can be repurposed to improve hematopoietic stem cell transplantation. Murine and human HSPCs were isolated from bone marrow and umbilical cord blood, respectively. Prostanoid receptor agonists and the combination thereof with forskolin were tested for their capacity to stimulate [(3)H]cAMP accumulation in HSPCs. Three independent approaches were employed to verify the ability of agonist-activated HSPCs to reconstitute the bone marrow in lethally irradiated recipient mice. The underlying mechanism was explored in cellular migration assays and by blocking C-X-C motif chemokine receptor 4 (CXCR4). Among several prostanoid agonists tested in combination with forskolin, treprostinil was most efficacious in raising intracellular cAMP levels in murine and human HPSCs. Injection of murine and human HSPCs, which had been pretreated with treprostinil and forskolin, enhanced survival of lethally irradiated recipient mice. Survival was further improved if recipient mice were subcutaneously administered treprostinil (0.15 mg kg(-1) 8 h(-1)) for 10 days. This regimen also reduced the number of HSPCs required to rescue lethally irradiated mice. Enhanced survival of recipient mice was causally related to treprostinil-enhanced CXCR4-dependent migration of HSPCs. Treprostinil stimulates the engraftment of human and murine hematopoietic stem cells without impairing their capacity for self-renewal. The investigated dose range corresponds to the dose approved for human use. Hence, these findings may be readily translated into a clinical application.

Personalizing health care: feasibility and future implications.

Considerable variety in how patients respond to treatments, driven by differences in their geno- and/ or phenotypes, calls for a more tailored approach. This is already happening, and will accelerate with developments in personalized medicine. However, its promise has not always translated into improvements in patient care due to the complexities involved. There are also concerns that advice for tests has been reversed, current tests can be costly, there is fragmentation of funding of care, and companies may seek high prices for new targeted drugs. There is a need to integrate current knowledge from a payer's perspective to provide future guidance. Multiple findings including general considerations; influence of pharmacogenomics on response and toxicity of drug therapies; value of biomarker tests; limitations and costs of tests; and potentially high acquisition costs of new targeted therapies help to give guidance on potential ways forward for all stakeholder groups. Overall, personalized medicine has the potential to revolutionize care. However, current challenges and concerns need to be addressed to enhance its uptake and funding to benefit patients.

CDK8-mediated STAT1-S727 phosphorylation restrains NK cell cytotoxicity and tumor surveillance.

The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance.

Targeting PI3Kδ: One man's meat is another man's poison.

We have recently uncovered the indispensable role of phosphoinositide-3-kinase δ (PI3Kδ) at different stages of the canonical killing pathway of cytotoxic T lymphocytes (CTLs). The interception of PI3Kδ-conveyed signals has been considered a valuable therapeutic strategy in oncology. However, our observations predict that the benefits of this approach may be limited by a trade-off between direct anticancer effects and an impaired ability of CTLs and NK cells to attack tumor cells.

Conditional IFNAR1 ablation reveals distinct requirements of Type I IFN signaling for NK cell maturation and tumor surveillance.

Mice with an impaired Type I interferon (IFN) signaling (IFNAR1- and IFNß-deficient mice) display an increased susceptibility toward v-ABL-induced B-cell leukemia/lymphoma. The enhanced leukemogenesis in the absence of an intact Type I IFN signaling is caused by alterations within the tumor environment. Deletion of Ifnar1 in tumor cells (as obtained in Ifnar1(f/f) CD19-Cre mice) failed to impact on disease latency or type. In line with this observation, the initial transformation and proliferative capacity of tumor cells were unaltered irrespective of whether the cells expressed IFNAR1 or not. v-ABL-induced leukemogenesis is mainly subjected to natural killer (NK) cell-mediated tumor surveillance. Thus, we concentrated on NK cell functions in IFNAR1 deficient animals. Ifnar1(-/-) NK cells displayed maturation defects as well as an impaired cytolytic activity. When we deleted Ifnar1 selectively in mature NK cells (by crossing Ncr1-iCre mice to Ifnar1(f/f) animals), maturation was not altered. However, NK cells derived from Ifnar1(f/f) Ncr1-iCre mice showed a significant cytolytic defect in vitro against the hematopoietic cell lines YAC-1 and RMA-S, but not against the melanoma cell line B16F10. Interestingly, this defect was not related to an in vivo phenotype as v-ABL-induced leukemogenesis was unaltered in Ifnar1(f/f )Ncr1-iCre compared with Ifnar1(f/f) control mice. Moreover, the ability of Ifnar1(f/f) Ncr1-iCre NK cells to kill B16F10 melanoma cells was unaltered, both in vitro and in vivo. Our data reveal that despite the necessity for Type I IFN in NK cell maturation the expression of IFNAR1 on mature murine NK cells is not required for efficient tumor surveillance.

PI3Kδ is essential for tumor clearance mediated by cytotoxic T lymphocytes.

BACKGROUND: PI3Kδ is a lipid kinase of the phosphoinositide 3-kinase class 1A family and involved in early signaling events of leukocytes regulating proliferation, differentiation and survival. Currently, several inhibitors of PI3Kδ are under investigation for the treatment of hematopoietic malignancies. In contrast to the beneficial effect of inhibiting PI3Kδ in tumor cells, several studies reported the requirement of PI3Kδ for the function of immune cells, such as natural killer and T helper cells. Cytotoxic T lymphocytes (CTLs) are essential for tumor surveillance. The scope of this study is to clarify the potential impact of PI3Kδ inhibition on the function of CTLs with emphasis on tumor surveillance. PRINCIPAL FINDINGS: PI3Kδ-deficient mice develop significantly bigger tumors when challenged with MC38 colon adenocarcinoma cells. This defect is accounted for by the fact that PI3Kδ controls the secretory perforin-granzyme pathway as well as the death-receptor pathway of CTL-mediated cytotoxicity, leading to severely diminished cytotoxicity against target cells in vitro and in vivo in the absence of PI3Kδ expression. PI3Kδ-deficient CTLs express low mRNA levels of important components of the cytotoxic machinery, e.g. prf1, grzmA, grzmB, fasl and trail. Accordingly, PI3Kδ-deficient tumor-infiltrating CTLs display a phenotype reminiscent of naïve T cells (CD69(low)CD62L(high)). In addition, electrophysiological capacitance measurements confirmed a fundamental degranulation defect of PI3Kδ-/- CTLs. CONCLUSION: Our results demonstrate that CTL-mediated tumor surveillance is severely impaired in the absence of PI3Kδ and predict that impaired immunosurveillance may limit the effectiveness of PI3Kδ inhibitors in long-term treatment.

c-JUN promotes BCR-ABL-induced lymphoid leukemia by inhibiting methylation of the 5' region of Cdk6.

The transcription factor c-JUN and its upstream kinase JNK1 have been implicated in BCR-ABL-induced leukemogenesis. JNK1 has been shown to regulate BCL2 expression, thereby altering leukemogenesis, but the impact of c-JUN remained unclear. In this study, we show that JNK1 and c-JUN promote leukemogenesis via separate pathways, because lack of c-JUN impairs proliferation of p185(BCR-ABL)-transformed cells without affecting their viability. The decreased proliferation of c-Jun(Δ/Δ) cells is associated with the loss of cyclin-dependent kinase 6 (CDK6) expression. In c-Jun(Δ/Δ) cells, CDK6 expression becomes down-regulated upon BCR-ABL-induced transformation, which correlates with CpG island methylation within the 5' region of Cdk6. We verified the impact of Cdk6 deficiency using Cdk6(-/-) mice that developed BCR-ABL-induced B-lymphoid leukemia with significantly increased latency and an attenuated disease phenotype. In addition, we show that reexpression of CDK6 in BCR-ABL-transformed c-Jun(Δ/Δ) cells reconstitutes proliferation and tumor formation in Nu/Nu mice. In summary, our study reveals a novel function for the activating protein 1 (AP-1) transcription factor c-JUN in leukemogenesis by antagonizing promoter methylation. Moreover, we identify CDK6 as relevant and critical target of AP-1-regulated DNA methylation on BCR-ABL-induced transformation, thereby accelerating leukemogenesis.