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Simultaneous inhibition of multiple kinases has been suggested to provide synergistic effects on inhibition of tumour growth and resistance. This study describes the design, synthesis and evaluation of 18 compounds incorporating a pyrrolo[2,3-d]pyrimidine scaffold for dual inhibition of epidermal growth factor receptor kinase (EGFR) and aurora kinase A (AURKA). Compounds 1–18 of this study demonstrate nanomolar inhibition of EGFR and micromolar inhibition of AURKA. Compounds 1–18 allow for a structure–activity relationships (SAR) analysis of the 4-anilino moiety for dual EGFR and AURKA inhibition. Compound 6, a 4-methoxyphenylpyrrolo[2,3-d]pyrimidin-4-amine, demonstrates single-digit micromolar inhibition of both AURKA and EGFR and provides evidence of a single molecule with dual activity against EGFR and AURKA. Compound 2, the most potent inhibitor of EGFR and AURKA from this series, has been further evaluated in four different squamous cell head and neck cancer cell lines for downstream effects resulting from AURKA and EGFR inhibition.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by complement-mediated hemolysis and thrombosis, and bone marrow failure. Affected cells harbor somatic mutation in X-linkedPIGAgene, essential for the initial step in glycosylphosphatidylinositol (GPI) biosynthesis. Loss of GPI biosynthesis results in defective cell-surface expression of GPI-anchored complement regulators CD59 and DAF. The affected stem cells generate many abnormal blood cells after clonal expansion, which occurs under bone marrow failure. Here, we report the mechanistic basis of a disease entity, autoinflammation-paroxysmal nocturnal hemoglobinuria (AIF-PNH), caused by germline mutation plus somatic loss ofPIGTon chromosome 20q. A region containing maternally imprinted genes implicated in clonal expansion in 20q-myeloproliferative syndromes was lost together with normalPIGTfrom paternal chromosome 20. Taking these findings together with a lack of bone marrow failure, the mechanisms of clonal expansion in AIF-PNH appear to differ from those in PNH. AIF-PNH is characterized by intravascular hemolysis and recurrent autoinflammation, such as urticaria, arthralgia, fever and aseptic meningitis. Consistent with PIGT’s essential role in synthesized GPI’s attachment to precursor proteins, non-protein-linked free GPIs appeared on the surface of PIGT-defective cells. PIGT-defective THP-1 cells accumulated higher levels of C3 fragments and C5b-9 complexes, and secreted more IL-1β than PIGA-defective cells after activation of the complement alternative pathway. IL-1β secretion was dependent upon C5b-9 complexes, accounting for the effectiveness of the anti-C5 drug eculizumab for both intravascular hemolysis and autoinflammation. These results suggest that free GPIs enhance complement activation and inflammasome-mediated IL-1β secretion.

Cyclin-dependent kinase subunit 1 (Cks1) is a critical rate-limiting component of the Skp1-Cullin1-Skp2 (SCF(Skp2)) ubiquitin ligase that controls cell cycle inhibitor abundance. Cyclin-dependent kinase (Cdk) inhibitors (CKIs) regulate hematopoietic stem cell (HSC) self-renewal, regeneration after cytotoxic stress and tumor cell proliferation. We thus studied the role of Cks1 in HSC and in a prototypic stem cell disorder, chronic myeloid leukemia (CML). Cks1 transcript was highly expressed in Lin-Sca-1+Kit+ (LSK) HSC, and the loss resulted in accumulation of the SCF(Skp2)/Cks1 substrates p21, p27, p57 and p130 particularly in CD150+ LSK cells. This accumulation correlated with decreased proliferation and accumulation of Cks1(-/-) HSC, slower regeneration after stress and prolonged HSC quiescence. At the hematopoietic progenitor (HPC) level, loss of Cks1 sensitized towards apoptosis. In CML, Cks1 expression was increased, and treatment with the Abl kinase inhibitor, imatinib, reduced Cks1 expression. Also, we found that Cks1 is critical for Bcr-Abl-induced cytokine-independent clonogenic activity. In conclusion, our study presents a novel function of Cks1 in maintaining HSC/HPC homeostasis and shows that Cks1 is a possible target in therapies aimed at the SCF(Skp2)/Cks1 complex that controls CKI abundance and cancer cell proliferation.

To ascertain the genetic basis of a paroxysmal nocturnal hemoglobinuria (PNH) case without somatic mutations in PIGA, we performed deep next-generation sequencing on all exons of known genes of the glycosylphosphatidylinositol (GPI) anchor synthesis pathway. We identified a heterozygous germline splice site mutation in PIGT and a somatic 8-MB deletion in granulocytes affecting the other copy of PIGT. PIGA is essential for GPI anchor synthesis, whereas PIGT is essential for attachment of the preassembled GPI anchor to proteins. Although a single mutation event in the X-chromosomal gene PIGA is known to cause GPI-anchored protein deficiency, 2 such hits are required in the autosomal gene PIGT. Our data indicate that PNH can occur even in the presence of fully assembled GPI if its transfer to proteins is defective in hematopoietic stem cells.

Cks1 is an activator of the SCF(Skp2) ubiquitin ligase complex that targets the cell cycle inhibitor p27(Kip1) for degradation. The loss of Cks1 results in p27(Kip1) accumulation and decreased proliferation and inhibits tumorigenesis. We identify here a function of Cks1 in mammalian cell cycle regulation that is independent of p27(Kip1). Specifically, Cks1(-/-); p27(Kip1-/-) mouse embryonic fibroblasts retain defects in the G(1)-S phase transition that are coupled with decreased Cdk2-associated kinase activity and defects in proliferation that are associated with Cks1 loss. Furthermore, concomitant loss of Cks1 does not rescue the tumor suppressor function of p27(Kip1) that is manifest in various organs of p27(Kip1-/-) mice. In contrast, defects in mitotic entry and premature senescence manifest in Cks1(-/-) cells are p27(Kip1) dependent. Collectively, these findings establish p27(Kip1)-independent functions of Cks1 in regulating the G(1)-S transition.

Alexander Hoellein 1 , Anja Pickhard 2 , Fabienne von Keitz 1 , Stephanie Schoeffmann 1 , Guido Piontek 2 , Martina Rudelius 3 , Anja Baumgart 1 , Stefan Wagenpfeil 4 , Christian Peschel 1 , Tobias Dechow 1 , Henning Bier 2 and Ulrich Keller 1 1 III. Medical Department, Technische Universitat Munchen, Munich, Germany 2 Department of Head and Neck Surgery, Technische Universitat Munchen, Munich, Germany 3 Institute of Pathology, Technische Universitat Munchen, Munich, Germany 4 Institute for Medical Statistics and Epidemiology, Technische Universitat Munchen, Munich, Germany Received: July 18, 2011; Accepted: August 22, 2011; Published: August 23, 2011; Keywords: Squamous cell cancer of the head and neck, Aurora kinase, EGFR Correspondence: Ulrich Keller, email: // // Abstract Squamous cell cancer of the head and neck (SCCHN) is the sixth leading cause for cancer deaths worldwide. Despite extense knowledge of risk factors and pathogenesis about 50 percent of all patients and essentially every patient with metastatic SCCHN eventually die from this disease. We analyzed the clinical data and performed immunohistochemistry for Epidermal growth factor receptor (EGFR) and Aurora kinase A (Aurora-A) expression in 180 SCCHN patients. Patients characterized by elevated EGFR and elevated Aurora-A protein expression in tumor tissue represent a risk group with poor disease-free and overall survival (EGFR low Aurora-A low versus EGFR high Aurora-A high , p=0.024). Treating SCCHN cell lines with a pan-Aurora kinase inhibitor resulted in defective cytokinesis, polyploidy and apoptosis, which was effective irrespective of the EGFR status. Combined Aurora kinase and EGFR targeting using a monoclonal anti-EGFR antibody was more effective compared to single EGFR and Aurora kinase inhibition. Comparing pan-Aurora kinase and Aurora-A targeting hints towards a strong and clinically relevant biological effect mediated via Aurora kinase B. Taken together, our findings characterize a new poor risk group in SCCHN patients defined by elevated EGFR and Aurora-A protein expression. Our results demonstrate that combined targeting of EGFR and Aurora kinases represents a therapeutic means to activate cell cycle checkpoints and apoptosis in SCCHN.

Myc oncoproteins promote continuous cell growth, in part by controlling the transcription of key cell cycle regulators. Here, we report that c-Myc regulates the expression of Aurora A and B kinases (Aurka and Aurkb), and that Aurka and Aurkb transcripts and protein levels are highly elevated in Myc-driven B-cell lymphomas in both mice and humans. The induction of Aurka by Myc is transcriptional and is directly mediated via E-boxes, whereas Aurkb is regulated indirectly. Blocking Aurka/b kinase activity with a selective Aurora kinase inhibitor triggers transient mitotic arrest, polyploidization, and apoptosis of Myc-induced lymphomas. These phenotypes are selectively bypassed by a kinase inhibitor-resistant Aurkb mutant, demonstrating that Aurkb is the primary therapeutic target in the context of Myc. Importantly, apoptosis provoked by Aurk inhibition was p53 independent, suggesting that Aurka/Aurkb inhibitors will show efficacy in treating primary or relapsed malignancies having Myc involvement and/or loss of p53 function.

Human metapneumovirus (hMPV) is an important cause of lower respiratory tract infection. In healthy subjects infections are usually mild and rarely necessitate hospitalization. However, more serious outcomes have been described for allogeneic stem cell transplant recipients. This study reports an outbreak of hMPV A2 infection in severely immunocompromised adult hematologic cancer patients in a tertiary care unit. HMPV RNA was detected in bronchoalveolar lavage or produced sputum from patients presenting with typical clinical features. A total of 15 patients were diagnosed in a period of 7 weeks. Molecular subtyping revealed infection with genotype A2a virus, implicating nosocomial transmission. Eleven patients (73%) were treated with intravenous immunoglobulins and ribavirin. Ten patients (65%) presented with severe dyspnea, five (33%) required mechanical ventilation. Four patients (26.6%) died from hMPV-associated pneumonia and consequent multi-organ failure. Thus, hMPV is a critical pathogen for patients with hematologic cancers warranting early detection.

The Cks1 component of the SCF(Skp2) complex is necessary for p27(Kip1) ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27(Kip1) levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27(Kip1) levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27(Kip1). To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo.

The universal cyclin-dependent kinase inhibitor p27Kip1 functions as a tumor suppressor, and reduced levels of p27Kip1 connote poor prognosis in several human malignancies. p27Kip1 levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCFSkp2 complex following its phosphorylation by the cyclin E–cyclin-dependent kinase 2 complex. Binding of phospho-p27Kip1 is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Eμ-Myc transgenic mice triggers p27Kip1 destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Eμ-Myc lymphomas and of human Burkitt lymphoma that bear MYC/Immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27Kip1 was abolished in Skp2-null Eμ-Myc B cells. However, the effect of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared with the effects of Cks1 loss. Collectively, these findings suggest that Cks1 targets, in addition to p27Kip1, are critical for Myc-driven proliferation and tumorigenesis. Mol Cancer Res; 8(3); 353–62

To assess whether the poor prognosis of ZAP70-positive B-cell chronic lymphocytic leukemia (CLL) is associated with the overexpression of ABC transporter genes that are responsible for pleiotropic drug resistance. The transcript level of ten drug transporters was analyzed using semiquantitative and quantitative RT-PCR in control hematopoietic cells, in 41 CLL patient samples and in 5 lymphoma cell lines. ZAP70 status was determined by immunoblotting. Of all analyzed transporters, MDR1, MDR2, MRP1, MRP4, MRP5, and MRP7 were expressed at a significantly higher level in B lymphocytes when compared with other hematopoietic cells in peripheral blood. A subgroup of 41 CLL patient samples showed similar or higher expression of these genes than control B cells, and CLL cells exhibited high expression when compared with multiple lymphoma cell lines. No significant correlation between ZAP70 expression and ABC transporter expression was observed. The ZAP70 status is independent of the multidrug resistance phenotype in CLL.

Myc oncoproteins (c-Myc, N-Myc and L-Myc) are transcription factors that regulate cell growth, cell division and metabolism under physiologic conditions. Myc overexpression is a hallmark of Burkitt lymphoma (BL) harboring MYC/IG translocations, and is frequently present in many advanced cancers. Myc overexpression is associated with aggressive disease, which is in part due to the destruction of select targets by the ubiquitin-proteasome system, for example Skp2SCF-directed destruction of the Cdk inhibitior p27Kip1 (Keller et al., EMBO 2007, Old et al., Mol. Canc. Res. 2010). We have identified a related means of post-translational protein modification, SUMOylation, as a pathway activated by Myc. Consequently, Myc-driven human BL and manifest Eµ-Myc mouse lymphomas are characterized by a hyperSUMOylation phenotype. Targeting SUMOylation in such lymphomas by a genetic approach results in growth inhibition and blocks lymphoma maintenance in vitro and in vivo. We further found that the SUMO pathway is in particular required for intact transitioning of the G2-M cell cycle checkpoint and through mitosis. The use of pharmacologic SUMOylation inhibitors (SUMOi) accordingly resulted in cell cycle arrest, polyploidy and cell death (Hoellein et al., Blood 2014). In this current report we aimed to identify crucial players of the Myc-SUMOylation axis by applying a mass spectrometry approach that used purified SUMOylated proteins in a conditional Myc-inducible B cell model. Stable isotope labeling permitted quantitative analysis of protein SUMOylation in the Myc-on versus Myc-off state. This screen identified Aurora kinase A as a Myc-regulated target of SUMOylation. Aurora kinases are essential regulators of mitosis and cytokinesis that are indispensable for Myc-driven transformed cells, and inhibition of Aurora kinase function results in similar effects as blocking SUMOylation, namely G2-M arrest, impaired cytokinesis resulting in polyploidy, and apoptosis (den Hollander et al., Blood 2010). We confirmed Myc-specific SUMO modification of Aurora kinase A and B and found reduced SUMOylation of both kinases upon treatment with pharmacologic SUMOylation inhibitors or shRNA targeting the key SUMOylation enzymes. Overexpression of Aurora kinase A and B mutants harboring a mutated SUMOylation motif resulted in a phenotype reminiscent of pharmacological SUMOi. This phenotype is independent of the Aurora kinase function since the SUMO motif mutation does not impair kinase activity. Moreover, in a comprehensive screen for specific E3-ligases that regulate the SUMO modification of the Aurora kinases we identified several SUMO E3 ligases that are also Myc induced. In summary, we propose a Myc-Aurora-kinase-SUMOylation circuit where Myc activation allows the induction of Aurora kinase transcription as well as the transcription of critical SUMOylation pathway genes. This mechanism contributes to sufficient Aurora kinase SUMOylation allowing faithful cell cycle passage and cytokinesis. In lymphoma and possibly other Myc-dependent cancers this pathway represents a target for synthetic lethal drug applications. Disclosures No relevant conflicts of interest to declare.

Myc oncoproteins (c-Myc, N-Myc and L-Myc) are transcription factors that regulate cell growth, cell division and metabolism under physiologic conditions. Myc overexpression is a hallmark of cancer, present in most advanced tumors, and associated with poor prognosis. We have previously shown that Myc overexpression results in specific cancer cell liabilities, e.g. during cell cycle progression, that constitute therapeutic targets for synthetic lethality approaches. Small Ubiquitin-like Modifier (SUMO) proteins covalently bind to other proteins to modify their function. SUMOylation is involved in various cellular processes including transcription and cell cycle progression. Hierarchical cluster analysis comparing RNA expression data in murine normal control, pre-malignant and lymphoma Eµ-Myc B cells identified a Myc-induced SUMOylation-related gene expression signature. This signature was present in pre-malignant and Eµ-Myc lymphoma cells and involved the up-regulation of various critical components of the SUMOylation machinery, including the E1 ligases SAE1 and SAE2, the E2 ligase Ube2i and the E3 ligases Ranbp2 and PIAS2. Moreover this translated into elevated protein expression of the whole SUMOylation pathway and ubiquitous hyper-SUMOylation of proteins in Eµ-Myc lymphoma cells. For cross-species validation we analyzed human gene expression data and found that the Myc-induced regulation of SUMOylation-associated genes was also present in human IG/MYC Burkitt lymphomas, in contrast to Non-IG/MYC B-cell lymphomas. What is more analysis of ChIP-on-chip experiments revealed direct binding of Myc to regulatory genomic regions of almost all SUMOylation regulators (SUMO2, SUMO3 and E1, E2 and E3 ligases). The characteristic of cancer cells to depend on certain intact physiologic mechanisms is known as non-oncogene addiction. Since SUMOylation of proteins is involved in essential metabolic, survival and proliferation pathways we reasoned that intact SUMOylation is a non-oncogenic pathway that Myc-driven cells rely upon. We thus hypothesized that Myc-dependent cells could be specifically susceptible when interfering with SUMOylation by pharmacological means. We found that Eµ-Myc lymphoma cells were highly sensitive to the SUMOylation inhibitors ginkolic acid and anarcardic acid. In particular, inhibition of SUMOylation lead to cell cycle arrest, polyploidy, and subsequent cell death. This therapeutic effect was Myc-specific as shown by use of genetically defined cell lines and conditional Myc-overexpression systems. Specifically human Burkitt lymphoma cell lines were strikingly more sensitive to inhibition of SUMOylation than non-Myc-transformed lymphoma samples. Taken together, we provide correlative and experimental evidence that the Myc-associated expression of genes involved in SUMOylation is a hallmark of Burkitt’s lymphoma and constitutes a non oncogenic pathway which is therapeutically exploitable in lymphoma and other Myc-driven cancers. Disclosures: No relevant conflicts of interest to declare.

Abstract 1328 Cks1 is an activator of the SCFSkp2 ubiquitin ligase complex that targets the cell cycle inhibitor p27Kip1 for degradation. Loss of Cks1 results in p27Kip1 accumulation and decreased proliferation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. Analysis of Cks1−/− murine embryonic fibroblasts (MEFs) unveiled a significant growth defect and comparison with double knock out Cks1−/−;p27Kip1−/− MEFs revealed a p27Kip1 independent defect at G1/S transition. These effects were associated with suppressed Cdk1 protein and Cdk2 activity in Cks1−/− cells. We hypothesized that Cks1 recruits further cell cycle regulators or facilitators of proliferation, which may explain the dramatic growth defect and the prolonged latency of lymphomagenesis upon loss of Cks1. Using Strep-Flag tagged Cks1 and tandem affinity purification followed by mass spectrometry we identified Geminin as a new Cks1 binding partner. Geminin is an inhibitor of rereplication and loss of Geminin leads to recurrent origin firing in S phase. The physical interaction of Cks1 and Geminin was present in cells of human and murine origin and was associated with Cdk2 binding to Geminin. Detailed analysis proved Cks1 dispensibel for cell cycle controlled Geminin degradation or phosphorylation. Surprisingly when mining public databases and RNA microarray data we found Geminin overexpressend in aggressive human lymphoma subtypes associated with high Ki-67 expression, and in murine Myc-induced lymphoma. Real time PCR revealed increased expression of Geminin during lymphomagenesis in Eμ-Myc mice, which correlated with protein expression. In conclusion, we established the physical Geminin-Cks1 protein interaction, present in human and murine cells, which could act in control of rereplication and genomic stability. Geminin is overexpressed in human and murine lymphoma revealing a potential role in lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.