From oncogenic replication stress to drug resistance : F-box proteins as signalling hubs in cancer

Cancer arises from cells that acquire genetic and epigenetic changes during the course of a, sometimes decades-long, somatic evolutionary process. These changes result in deregulation of a multitude of cellular processes leading to novel capabilities, often referred to as hallmarks of cancer, and a strong selective advantage for these cells albeit at a dramatic cost to the organism as a whole. Both, gene expression but also turn-over of gene products can become deregulated. The ubiquitin-proteasome system is responsible for the targeted degradation of proteins, and components of this system are altered during cancer development. Target specificity of this system is largely attained through E3 ubiquitin ligases that mediate the covalent attachment of ubiquitin to their substrates. The largest group of E3s are cullin-RING domain ligases (CRLs) with SKP1-cullin1-F-box protein (SCF) E3 ligases, or CRL1, representing one of the best-characterised subgroups of CRLs. These SCF ligases are multiprotein complexes containing one of, in human cells, 69 F-box proteins which function as substrate-adaptor subunits. Collectively, the family of F-box proteins has been found to be critically involved in virtually all the cancer hallmarks. However, despite their important role in cancer development, only a handful of SCFs has been molecularly and functionally well- characterised and detailed knowledge of how deregulation of specific SCF ligases and downstream substrate effectors impinges on cancer traits is lacking. One of the main aims of the work presented in this thesis is to find cellular vulnerabilities resulting from deregulation of F-box proteins in cancer. FBXW7 is the most commonly mutated F-box protein in human cancers. Its inactivation leads to upregulation of its substrates including cyclin E, MYC or SOX9 (paper IV) resulting in deregulated proliferation, increased metastasis and drug resistance but also replication stress. Cancer cells undergoing replication stress become more dependent on signalling pathways detecting and repairing damaged DNA (papers I and III) and are consequently more sensitive to therapies targeting checkpoint and repair proteins such as WEE1, ATR or DNA-PK kinases (paper II). In paper I we describe a novel function for the largely uncharacterised F-box protein FBXL12 in regulating the response to oncogene-induced replication stress. FBXL12 complements the Fanconi anaemia (FA) DNA repair pathway by targeting its central component FANCD2 for proteasomal degradation. The FA pathway not only plays a crucial role in resilience to endogenous sources of replication stress but also to drug-induced stress. FBXL12 and cyclin E are upregulated and correlated in human cancers and depletion of FBXL12 results in increased sensitivity to replication stress which posits FBXL12 as a potential cancer drug target. Ablation or pharmacological inhibition of FBXL12 prevents degradation of FANCD2 and breast cancer cells are sensitised to the adverse effects of drug- as well as oncogenic cyclin E-induced replication stress. In paper II we focus on exploring further ways of sensitising cancer cells to replication stress. We performed a screen to identify potential viability markers in response to replication stress induced by WEE1 inhibitor AZD1775 and discover novel synergistic combinations. Additionally, we determine a subset of basal-like breast cancer cells that responds to treatment initially but recovers after treatment cessation and identify PTEN as a novel predictive marker for such responses, with cells expressing low levels of PTEN being highly sensitive acutely and failing to recover. Furthermore, inactivation or genomic deletion of DNA-PK, an apical DNA damage kinase, attenuates recovery and sensitises basal-like breast cancer cells to AZD1775. Mechanistically, loss of PTEN or DNA-PK impair CHK1 activation and S-phase arrest in response to AZD1775 treatment, which finally ensues lethal replication stress and loss of survival. In paper III we concentrate on FBXO28, another poorly-studied member of the F-box family, which we find to degrade ARHGEF6 and ARHGEF7 activators of the Rho-type GTPase RAC1, involved in cell motility. Surprisingly, we identify a novel function for FBXO28 and ARHGEF6/7 in promoting the repair of breaks in heterochromatin DNA. Following DNA damage, tightly chromatin-bound FBXO28 is released and promotes degradation of nucleoplasmic ARHGEF6/7 to modulate activation and inactivation cycles of nuclear RAC1 and allow for efficient resolution of H3K9me2/3-positive damaged sites. In paper IV we add a key oncogenic transcription factor to the growing list of FBXW7 substrates; SOX9. FBXW7 ubiquitylates and degrades SOX9 upon phosphorylation by GSK3β. Mutation and inactivation of FBXW7 in medulloblastoma concurs with elevated SOX9 protein expression and poor patient outcome. In medulloblastoma cell line models we demonstrate increased cell motility, metastasis and increased resistance to cytostatic treatment after expression of a non-degradable SOX9 mutant. Conversely, inhibition of the PI3K/AKT/mTOR pathway promoted GSK3β-dependent SOX9 degradation and sensitised FBXW7-proficient medulloblastoma cells to cisplatin

USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma

The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC‐induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X‐linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B‐cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event‐free survival in patients treated with spindle poison‐containing chemotherapy. Accordingly, aggressive B‐cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eμ‐Myc lymphoma model. Together, we specify the USP9X–XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B‐cell lymphoma

Characterization of YBCO films by torque magnetometry

We report on the observation of a thermopile effect caused by laser radiation heating in thin films of high-Tc material. The thermopile effect is due to a transverse Seebeck effect. The transverse Seebeck effect is observable first because of the anisotropy of YBa2Cu3O7−δ, and secondly because it is possible to grow off-axis epitaxial films. The thermopile effect can be used for developing detectors for laser radiation

Mutations in the Arabidopsis RPK1 gene uncouple cotyledon anlagen and primordia by modulating epidermal cell shape and polarity

Summary Plant seedlings have either one or two cotyledons. The mechanisms that regulate this organ number are poorly understood. Mutations in the RECEPTOR-LIKE PROTEIN KINASE1 (RPK1) gene of the dicot Arabidopsis have only one cotyledon, with low penetrance due to complex genetic redundancy. An analysis of patterning genes required for cotyledon initiation showed that these have normal expression patterns, defining the cotyledon anlagen, in rpk1. This was also true for key genes, which organize the shoot apical meristem (SAM). By contrast, epidermal cell shape and polarity were compromised in rpk1 embryos, as evidenced by disturbed polarity of the auxin efflux carrier PIN1. PIN1 is required for the establishment of auxin maxima, which induce and maintain organ primordia. The effects in rpk1 mutants manifest in a spatially and timely stochastic fashion probably due to redundancy of RPK1-like functions. Consistently, auxin maxima showed a stochastic distribution in rpk1 embryos, being at times entirely absent and at other times supernumerary. This variability may explain how monocotyledonous seedlings and cotyledon shape variants can developmentally arise in Arabidopsis and possibly in other plants

USP9X stabilizes XIAP to regulate mitotic cell death and chemoresistance in aggressive B-cell lymphoma

The mitotic spindle assembly checkpoint (SAC) maintains genome stability and marks an important target for antineoplastic therapies. However, it has remained unclear how cells execute cell fate decisions under conditions of SAC‐induced mitotic arrest. Here, we identify USP9X as the mitotic deubiquitinase of the X‐linked inhibitor of apoptosis protein (XIAP) and demonstrate that deubiquitylation and stabilization of XIAP by USP9X lead to increased resistance toward mitotic spindle poisons. We find that primary human aggressive B‐cell lymphoma samples exhibit high USP9X expression that correlate with XIAP overexpression. We show that high USP9X/XIAP expression is associated with shorter event‐free survival in patients treated with spindle poison‐containing chemotherapy. Accordingly, aggressive B‐cell lymphoma lines with USP9X and associated XIAP overexpression exhibit increased chemoresistance, reversed by specific inhibition of either USP9X or XIAP. Moreover, knockdown of USP9X or XIAP significantly delays lymphoma development and increases sensitivity to spindle poisons in a murine Eμ‐Myc lymphoma model. Together, we specify the USP9X–XIAP axis as a regulator of the mitotic cell fate decision and propose that USP9X and XIAP are potential prognostic biomarkers and therapeutic targets in aggressive B‐cell lymphoma

FBW7 suppression leads to SOX9 stabilization and increased malignancy in medulloblastoma

SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7 alpha. Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.Aldwin Suryo Rahmanto and Vasil Savov contributed equally to this work as first authorsAndrä Brunner, Sara Bolin and Holger Weishaupt contributed equally to this work as second authorsFredrik J Swartling and Olle Sangfelt contributed equally to this work as corresponding authors</p

Guidelines for the use of flow cytometry and cell sorting in immunological studies

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis