Michaela Prchal-Murphy

CDK6 as a key regulator of hematopoietic and leukemic stem cell activation.

The cyclin-dependent kinase 6 (CDK6) and CDK4 have redundant functions in regulating cell-cycle progression. We describe a novel role for CDK6 in hematopoietic and leukemic stem cells (hematopoietic stem cells [HSCs] and leukemic stem cells [LSCs]) that exceeds its function as a cell-cycle regulator. Although hematopoiesis appears normal under steady-state conditions, Cdk6(-/-) HSCs do not efficiently repopulate upon competitive transplantation, and Cdk6-deficient mice are significantly more susceptible to 5-fluorouracil treatment. We find that activation of HSCs requires CDK6, which interferes with the transcription of key regulators, including Egr1. Transcriptional profiling of HSCs is consistent with the central role of Egr1. The impaired repopulation capacity extends to BCR-ABL(p210+) LSCs. Transplantation with BCR-ABL(p210+)-infected bone marrow from Cdk6(-/-) mice fails to induce disease, although recipient mice do harbor LSCs. Egr1 knock-down in Cdk6(-/-) BCR-ABL(p210+) LSKs significantly enhances the potential to form colonies, underlining the importance of the CDK6-Egr1 axis. Our findings define CDK6 as an important regulator of stem cell activation and an essential component of a transcriptional complex that suppresses Egr1 in HSCs and LSCs.

STAT5 Is a Key Regulator in NK Cells and Acts as a Molecular Switch from Tumor Surveillance to Tumor Promotion

UNLABELLED Natural killer (NK) cells are tightly regulated by the JAK-STAT signaling pathway and cannot survive in the absence of STAT5. We now report that STAT5-deficient NK cells can be rescued by overexpression of BCL2. Our experiments define STAT5 as a master regulator of NK-cell proliferation and lytic functions. Although NK cells are generally responsible for killing tumor cells, the rescued STAT5-deficient NK cells promote tumor formation by producing enhanced levels of the angiogenic factor VEGFA. The importance of VEGFA produced by NK cells was verified by experiments with a conditional knockout of VEGFA in NK cells. We show that STAT5 normally represses the transcription of VEGFA in NK cells, in both mice and humans. These findings reveal that STAT5-directed therapies may have negative effects: In addition to impairing NK-cell-mediated tumor surveillance, they may even promote tumor growth by enhancing angiogenesis. SIGNIFICANCE The importance of the immune system in effective cancer treatment is widely recognized. We show that the new signal interceptors targeting the JAK-STAT5 pathway may have dangerous side effects that must be taken into account in clinical trials: inhibiting JAK-STAT5 has the potential to promote tumor growth by enhancing NK-cell-mediated angiogenesis.

TYK2 kinase activity is required for functional type I interferon responses in vivo.

Tyrosine kinase 2 (TYK2) is a member of the Janus kinase (JAK) family and is involved in cytokine signalling. In vitro analyses suggest that TYK2 also has kinase-independent, i.e., non-canonical, functions. We have generated gene-targeted mice harbouring a mutation in the ATP-binding pocket of the kinase domain. The Tyk2 kinase-inactive (Tyk2K923E) mice are viable and show no gross abnormalities. We show that kinase-active TYK2 is required for full-fledged type I interferon- (IFN) induced activation of the transcription factors STAT1-4 and for the in vivo antiviral defence against viruses primarily controlled through type I IFN actions. In addition, TYK2 kinase activity was found to be required for the protein’s stability. An inhibitory function was only observed upon over-expression of TYK2K923E in vitro. Tyk2K923E mice represent the first model for studying the kinase-independent function of a JAK in vivo and for assessing the consequences of side effects of JAK inhibitors.

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 (CD69lowCD62Lhigh). 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.

Palbociclib treatment of FLT3-ITD+ AML cells uncovers a kinase-dependent transcriptional regulation of FLT3 and PIM1 by CDK6

Up to 30% of patients with acute myeloid leukemia have constitutively activating internal tandem duplications (ITDs) of the FLT3 receptor tyrosine kinase. Such mutations are associated with a poor prognosis and a high propensity to relapse after remission. FLT3 inhibitors are being developed as targeted therapy for FLT3-ITD(+) acute myeloid leukemia; however, their use is complicated by rapid development of resistance, which illustrates the need for additional therapeutic targets. We show that the US Food and Drug Administration-approved CDK4/6 kinase inhibitor palbociclib induces apoptosis of FLT3-ITD leukemic cells. The effect is specific for FLT3-mutant cells and is ascribed to the transcriptional activity of CDK6: CDK6 but not its functional homolog CDK4 is found at the promoters of the FLT3 and PIM1 genes, another important leukemogenic driver. There CDK6 regulates transcription in a kinase-dependent manner. Of potential clinical relevance, combined treatment with palbociclib and FLT3 inhibitors results in synergistic cytotoxicity. Simultaneously targeting two critical signaling nodes in leukemogenesis could represent a therapeutic breakthrough, leading to complete remission and overcoming resistance to FLT3 inhibitors.

Cdk4 and Cdk6 cooperate in counteracting the INK4 family of inhibitors during murine leukemogenesis

Cdk4 and Cdk6 are related protein kinases that bind d-type cyclins and regulate cell-cycle progression. Cdk4/6 inhibitors are currently being used in advanced clinical trials and show great promise against many types of tumors. Cdk4 and Cdk6 are inhibited by INK4 proteins, which exert tumor-suppressing functions. To test the significance of this inhibitory mechanism, we generated knock-in mice that express a Cdk6 mutant (Cdk6 R31C) insensitive to INK4-mediated inhibition. Cdk6(R/R) mice display altered development of the hematopoietic system without enhanced tumor susceptibility, either in the presence or absence of p53. Unexpectedly, Cdk6 R31C impairs the potential of hematopoietic progenitors to repopulate upon adoptive transfer or after 5-fluorouracil-induced damage. The defects are overcome by eliminating sensitivity of cells to INK4 inhibitors by introducing the INK4-insensitive Cdk4 R24C allele, and INK4-resistant mice are more susceptible to hematopoietic and endocrine tumors. In BCR-ABL-transformed hematopoietic cells, Cdk6 R31C causes increased binding of p16(INK4a) to wild-type Cdk4, whereas cells harboring Cdk4 R24C and Cdk6 R31C are fully insensitive to INK4 inhibitors, resulting in accelerated disease onset. Our observations reveal that Cdk4 and Cdk6 cooperate in hematopoietic tumor development and suggest a role for Cdk6 in sequestering INK4 proteins away from Cdk4.

Dendritic cell-secreted lipocalin2 induces CD8+ T-cell apoptosis, contributes to T-cell priming and leads to a TH1 phenotype.

Lipocalin 2 (LCN2), which is highly expressed by dendritic cells (DCs) when treated with dexamethasone (Dex) and lipopolysaccharide (LPS), plays a key role in the defence against bacteria and is also involved in the autocrine apoptosis of T-cells. However, the function of LCN2 when secreted by DCs is unknown: this is a critical gap in our understanding of the regulation of innate and adaptive immune systems. Tolerance, stimulation and suppression are functions of DCs that facilitate the fine-tuning of the immune responses and which are possibly influenced by LCN2 secretion. We therefore examined the role of LCN2 in DC/T-cell interaction. WT or Lcn2−/− bone marrow-derived DCs were stimulated with LPS or LPS+IFN-γ with and without Dex and subsequently co-cultured with T-cells from ovalbumin-specific TCR transgenic (OT-I and OT-II) mice. We found that CD8+ T-cell apoptosis was highly reduced when Lcn2−/− DCs were compared with WT. An in vivo CTL assay, using LPS-treated DCs, showed diminished killing ability in mice that had received Lcn2−/− DCs compared with WT DCs. As a consequence, we analysed T-cell proliferation and found that LCN2 participates in T-cell-priming in a dose-dependent manner and promotes a TH1 microenvironment. DC-secreted LCN2, whose function has previously been unknown, may in fact have an important role in regulating the balance between TH1 and TH2. Our results yield insights into DC-secreted LCN2 activity, which could play a pivotal role in cellular immune therapy and in regulating immune responses.

In vivo tumor surveillance by NK cells requires TYK2 but not TYK2 kinase activity

Tyrosine kinase 2 (TYK2) is a Janus kinase (JAK) that is crucially involved in inflammation, carcinogenesis and defense against infection. The cytotoxic activity of natural killer (NK) cells in TYK2-deficient (Tyk2−/−) mice is severely reduced, although the underlying mechanisms are largely unknown. Using Tyk2−/− mice and mice expressing a kinase-inactive version of TYK2 (Tyk2K923E), we show that NK cell function is partly independent of the enzymatic activity of TYK2. Tyk2−/− and Tyk2K923E NK cells develop normally in the bone marrow, but the maturation of splenic Tyk2−/− NK cells (and to a lesser extent of Tyk2K923E NK cells) is impaired. In contrast, the production of interferon γ (IFNγ) in response to interleukin 12 (IL-12) or to stimulation through NK cell-activating receptors strictly depends on the presence of enzymatically active TYK2. The cytotoxic activity of Tyk2K923E NK cells against a range of target cells in vitro is higher than that of Tyk2−/− NK cells. Consistently, Tyk2K923E mice control the growth of NK cell-targeted tumors significantly better than TYK2-deficient mice, showing the physiological relevance of the finding. Inhibitors of TYK2s kinase activity are being developed for the treatment of inflammatory diseases and cancers, but their effects on tumor immune surveillance have not been investigated. Our finding that TYK2 has kinase-independent functions in vivo suggests that such inhibitors will leave NK cell mediated tumor surveillance largely intact and that they will be suitable for use in cancer therapy.

Stat5 gene dosage in T cells modulates CD8+ T‐cell homeostasis and attenuates contact hypersensitivity response in mice

Contact hypersensitivity assay (CHS) faithfully models human allergies. The Stat5 transcription factors are essential for both lymphocyte development and acute immune responses. Although consequences of Stat5 ablation and transgenic overexpression for the lymphocyte development and functions have been extensively studied, the role of Stat5 gene dosage in contact allergies has not been addressed.

STAT5BN642H is a driver mutation for T cell neoplasia

STAT5B is often mutated in hematopoietic malignancies. The most frequent STAT5B mutation, Asp642His (N642H), has been found in over 90 leukemia and lymphoma patients. Here, we used the Vav1 promoter to generate transgenic mouse models that expressed either human STAT5B or STAT5BN642H in the hematopoietic compartment. While STAT5B-expressing mice lacked a hematopoietic phenotype, the STAT5BN642H-expressing mice rapidly developed T cell neoplasms. Neoplasia manifested as transplantable CD8+ lymphoma or leukemia, indicating that the STAT5BN642H mutation drives cancer development. Persistent and enhanced levels of STAT5BN642H tyrosine phosphorylation in transformed CD8+ T cells led to profound changes in gene expression that were accompanied by alterations in DNA methylation at potential histone methyltransferase EZH2-binding sites. Aurora kinase genes were enriched in STAT5BN642H-expressing CD8+ T cells, which were exquisitely sensitive to JAK and Aurora kinase inhibitors. Together, our data suggest that JAK and Aurora kinase inhibitors should be further explored as potential therapeutics for lymphoma and leukemia patients with the STAT5BN642H mutation who respond poorly to conventional chemotherapy.