CDK6 in lymphoid malignancy and disease

Cyclin abhängige Kinasen (CDKs) spielen eine wichtige Rolle in der Regulierung des Zellzyklus. Eine Deregulierung dieser Zellzykluskinasen ist häufig mit unkontrolliertem Zellwachstum und Tumorbildung vergesellschaftet. Die Hemmung der CDKs durch Signal-Interzeptoren zur Therapie maligner Tumore ist Gegenstand intensiver klinischer Forschung. CDK6, eine Kinase der G1-Phase, ist besonders stark in lymphoiden Tumoren exprimiert. Aus diesem Grund untersuchten wir die Bedeutung von CDK6 für p185BCR-ABL und NPM-ALK-induzierte Tumorgenese. Unerwarteterweise kann eine erhöhte CDK6 Aktivität Tumorwachstum sowohl fördern als auch hemmen. Die Leukämieentwicklung in Cdk6-/- Mäusen war stark verzögert, dies korrelierte mit einer reduzierten Proliferation der transformierten lymphoiden Zellen. Ein ähnliches Resultat, eine reduzierte Proliferation, erzielten wir aber auch durch die erhöhte Expression von CDK6. p185BCR-ABL-transformierte Zellen mit erhöhter CDK6 Expression induzierten Leukämie/Lymphome mit verringerter Inzidenz und verlängerter Latenz. Diese unerwartete Tumor unterdrückende Eigenschaft von CDK6 liegt einer transkriptionellen Aktivität der Kinase zu Grunde. CDK6 bindet an den Promoterbereich des Tumorsuppressors, p16INK4a und fördert somit dessen Expression in einer Kinase unabhängigen Weise. Für diesen Effekt spielt der C-terminus von CDK6 eine entscheidende Rolle. Eine fehlende p16INK4a Expression ist daher die Voraussetzung, dass CDK6 die Tumorbildung beschleunigt. Studien in humanen B- und T-Zell Lymphomen bestätigte eine inverse Beziehung zwischen CDK6 und p16INK4a. Neben der Regulation von p16INK4a, ist CDK6 auch in die transkriptionelle Regulation des Pro-Angiogenese Faktors VEGF involviert. CDK6 defiziente leukämische Zellen wiesen reduzierte Vegf-A und -C mRNA Werte auf. ^Re-expression von CDK6 in Cdk6-/- leukämischen Zellen erhöhte die Vegf-A und -C mRNA Expression und führte zu schnellerem Wachstum und besserer Versorgung des Tumors. Diese hier erstmals beschriebene Rolle von CDK6 ist ebenfalls Kinase unabhängig, und wird durch den C-terminus mediiert. Weiters konnte ich feststellen, dass p185BCR-ABL-transformierte Zellen, denen der AP-1 Transkriptionsfaktor c-JUN fehlt, erniedrigte CDK6 und Vegf-A Werte haben. Dies war mit einer reduzierten Tumorbildung sowie einer verlangsamten Leukämieentwickling vergesellschaftet. Das Muster der deregulierten Angiogenese-steuernden Gene ist vergleichbar zwischen c-Jun[Delta]/[Delta] und Cdk6-/- leukämischen Zellen. Dies lässt eine Interaktion zwischen AP-1 Transkriptionsfaktoren und CDK6 vermuten. Ein potentieller therapeutischer Nutzen von CDK6 Inhibitoren soll mit dieser Studie für Patienten individuell bestimmt werden können. Unsere Studie zeigt nun zum erstenmal die Rolle von hohen CDK6 Werten in Patienten und das CDK6, auf Kinase unabhängige Weise, eine wichtige Rolle in der Regulation kritischer Faktoren in der Tumorentwicklung hat.Small molecular compounds directed against cell cycle kinases are currently developed and tested for their optimal clinical use to treat cancer in people. The kinase CDK6 is expressed at high levels in lymphoid malignancies. We therefore studied the impact of CDK6 for p185BCR-ABL and NPM-ALK-driven tumorigenesis and discovered a bistable outcome: enhanced CDK6 activity may either promote or delay tumor growth. As anticipated, leukemia/lymphoma formation was strongly delayed in Cdk6-/- mice due to a reduced proliferation of the transformed lymphoid cells. Surprisingly, a similar outcome was observed upon enforced CDK6 expression in p185BCR-ABL-transformed tumor cells. These cells induced lymphoma/leukemia with decreased incidence and increased latency. This unexpected tumor suppressing property of CDK6 was accounted to the finding that CDK6 bound to the promoter of the tumor suppressor, p16INK4a and caused its expression in a kinase independent fashion. The C-terminus of CDK6 plays a critical role in this transcription regulation. Hence, a tumor promoting effect of elevated CDK6 levels - as observed in lymphoid malignancies - required a disrupted p16INK4a expression. Studies of human B- and T-cell lymphomas confirmed an inverse relationship between CDK6 and p16INK4a. Beside acting as a tumor promoter or suppressor we detected, that CDK6 is involved in the transcriptional regulation of the pro-angiogenetic factor VEGF. Leukemic cells lacking CDK6 displayed significantly reduced Vegf-A and -C mRNA levels. The re-expression of CDK6 in Cdk6-/- cells re-established Vegf-A and -C secretion. Accordingly, tumor formation increased upon CDK6 re-expression. VEGF induction by CDK6 is independent of its kinase activity but requires the C-terminus. Moreover, we found that p185BCR-ABL-transformed cells lacking the AP-1 transcription factor c-JUN, fail to express CDK6. In vivo, these cells induced tumors and leukemia with increased latency. Importantly c-Jun[Delta]/[Delta] leukemic cells lacking CDK6 displayed comparable alterations in angiogenic pathway as Cdk6-/- leukemic cells. These findings suggest a potential interaction between AP-1 family members and CDK6 in leukemia. This insight is of major importance to anticipate and predict which patients will benefit from a treatment interfering with CDK6.applicant: Karoline KollmannAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2010OeBB(VLID)188340

CDK6 Inhibition: A Novel Approach in AML Management

Acute myeloid leukemia (AML) is a complex disease with an aggressive clinical course and high mortality rate. The standard of care for patients has only changed minimally over the past 40 years. However, potentially useful agents have moved from bench to bedside with the potential to revolutionize therapeutic strategies. As such, cell-cycle inhibitors have been discussed as alternative treatment options for AML. In this review, we focus on cyclin-dependent kinase 6 (CDK6) emerging as a key molecule with distinct functions in different subsets of AML. CDK6 exerts its effects in a kinase-dependent and -independent manner which is of clinical significance as current inhibitors only target the enzymatic activity

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.This work was supported by grants from the Austrian Science Foundation (FWF) (SFB47 and P24297) (V.S.), fellowships from the Spanish Ministerio de Economia y Competitividad (MINECO) (E.R.-D., V.Q.), and grants from MINECO (SAF2012-38215), Fundacion Ramon Areces, the OncoCycle Programme (S2010/BMD-2470) from the Comunidad de Madrid, the OncoBIO Consolider-Ingenio 2010 Programme (CSD2007-00017) from MINECO, and the European Union Seventh Framework Programme (MitoSys project; HEALTH-F5-2010-241548) (M.M.).S

A robust approach for the generation of functional hematopoietic progenitor cell lines to model leukemic transformation

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (Lin-, Sca-1+, c-Kit+) cells, which closely resemble MPP1 cells. HPCLSKs reconstitute hematopoiesis in lethally irradiated recipient mice over >8 months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD, or MLL-AF9, their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSKs can be applied to transgenic mice, and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, HPCLSKs Cdk6-/- induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells