Investigating mTORC2/AKT-mediated regulation of FOXO1: a novel therapeutic strategy for chronic lymphocytic leukaemia?

The pathogenesis of chronic lymphocytic leukaemia (CLL) is inextricably linked to the tumour microenvironment, a ‘sanctuary site’ wherein CLL cells engage B cell receptor (BCR) signalling and form interactions with non-malignant accessory cells. ‘Crosstalk’ within the microenvironment elicits survival and proliferative signals that facilitate therapy resistance and outgrowth of malignant clones. Therefore, eliminating the signals that orchestrate these events is crucial to prevent disease progression. The advent of small molecule inhibitors targeting BCR signalling components have proven clinically effective. However, these treatments are not always available (or indeed suitable) for every patient and drug resistance has been reported. Thus, there is a need to identify novel treatment strategies that have the ability to improve CLL patient outcomes. Several oncogenic pathways emanating from microenvironment communication converge upon the PI3K-AKT-mTOR axis in CLL cells. Surprisingly, little is known about the functional importance of mTOR signalling in CLL pathogenesis. mTOR exists in two protein complexes, mTORC1 and mTORC2, which coordinate growth, survival and proliferation signals downstream of PI3K-AKT signalling. Despite encouraging preclinical data with the mTORC1-selective inhibitor rapamycin, the rapalogue everolimus only had modest anti-tumour activity in a CLL clinical trial. Clinical activity of mTORC1-selective inhibitors is limited due to abrogation of a S6K-mediated negative feedback loop modulating mTORC2 activity, which results in activation of AKT-mediated pro-survival signalling. The development of ‘second generation’ ATP-competitive mTOR inhibitors avoid these issues by inhibiting both mTORC1 and mTORC2. As such, this investigation sought to address whether inhibition of mTORC1/2 with the dual mTOR kinase inhibitor AZD8055 would represent an effective therapeutic approach for CLL. The data presented in this thesis demonstrates that mTOR is an effector of BCR crosslinking in vitro, playing a role in the coordination cellular behaviours emanating from BCR engagement (BCR-PI3K-AKT) in CLL cells. mTORC1 (4E-BP1T37/46 and S6S235/236) and mTORC2 (AKTS473) activities were effectively targeted by the ‘second generation’ mTOR kinase inhibitor AZD8055 (and its clinical analogue AZD2014), which disabled pro-survival feedback loops associated with rapamycin treatment. At the molecular level, AZD8055 inhibited mTOR signalling downstream of F(ab’)2-mediated BCR ligation and stromal cell (NT-L/CD40L) co-cultures, highlighting the ability of this compound to disrupt various microenvironmental stimuli. On a functional level, AZD8055 elicited potent inhibitory effects on CLL growth and proliferation, but only moderately affected cell viability in vitro. For these reasons, AZD8055 anti-tumour activity appeared to be limited as a monotherapy. A synergistic combination of AZD8055 and the BTK inhibitor ibrutinib promoted cell death, augmented cell size contraction and arrested proliferation, indicating that simultaneous inhibition of mTOR kinase and BTK in CLL cells evokes anti-tumour activity via targeted inhibition of multiple oncogenic pathways and at different levels within the same pathway. In search of a mechanism of action, we proposed that the combination treatment conferred a more robust inhibition of AKT kinase activity. Among other methods, AKT promotes cell survival and proliferation via negative regulation of FOXO transcription factors. FOXOs are widely regarded as tumour suppressors, which regulate several cellular behaviours including cell cycle arrest and apoptosis. Our data demonstrated that BCR crosslinking negatively regulated FOXO1 (the most abundant FOXO in CLL cells) by AKT-dependent FOXO1T24 phosphorylation, subsequent nuclear export and reduced DNA-binding activity. Like normal B cells, these data suggested that FOXO1 inactivation was an important consequence of BCR engagement in CLL cells. For this reason, we hypothesised that inhibiting BCR signalling would unleash FOXO1 tumour suppressor activity. We showed that elimination of BCR signal transduction, via AZD8055 or ibrutinib mono- and combination therapy, re-engaged FOXO1 DNA-binding activity by preventing FOXO1 nuclear export, which suggested that FOXO1 was an effector of BCR signalling inhibition that mediated treatment response. Through pharmacological FOXO1 inhibition, we demonstrated that FOXO1 activity contributed to the cytotoxic, cell-contracting and cytostatic effects of the combination treatment, indicating that FOXO1 functions as a tumour suppressor in this context. In conclusion, these studies highlight the potential for AZD8055/2014 as a drug partner for novel combination strategies in the treatment of CLL. Indeed, these data suggest that AZD8055/2014 could be combined with agents targeting proximal BCR signalling components (BTK, PI3K or SYK) or perhaps pro-survival signals (BCL2). Furthermore, reactivation of FOXO1 activity or other inactivated ‘genetically intact’ tumour suppressors (via targeted inhibition of negative regulators) represents an interesting treatment strategy for CLL. Collectively, these data provide valuable information that has the potential to inform subsequent pre-clinical investigations and future CLL clinical trials

B-cell receptor signaling- and p53-dependent non-coding RNA expression in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in adults of the Western world. It is a malignancy characterized by an accumulation of CD5 positive B-cells in blood and lymphoid organs. CLL is a very heterogeneous disease, where molecular subgroups display striking differences in treatment response and prognosis. A greater BCR signaling capacity and a loss of p53 signaling activity confer a poor prognosis. While the higher BCR signaling activity seen in CLL with unmutated IGHV genes supports tumor cell survival, p53 aberrations mediate resistance towards standard therapy. The aim of this work was to characterize the involvement of non-coding RNA in these two key signaling pathways of CLL cell survival and resistance. Small RNA sequencing was applied to comprehensively assess microRNA (miRNA) and other non-coding RNA expression in peripheral blood mononuclear cells of 35 CLL patients. miRNAs were identified that display IGHV mutation status dependent expression, and the transcript levels of 15 miRNAs predicted IGHV mutation status with 82% accuracy. By abrogation of BCR signaling in vitro using the small-molecule inhibitor ibrutinib, the expression of miR-320c, miR-1246, miR-484, miR-17-5p, miR-155-3p and miR-27a-5p was found to be BCR signaling dependent, suggesting a role in mediating CLL cell survival. The basal expression of 10 miRNAs was associated with ibrutinib sensitivity in vitro, implicating an involvement of these miRNAs in the regulation of BCR signaling. It was hypothesized that p53-dependent ncRNAs could be identified by comparison of CLL samples with or without TP53 mutation/deletion for their ncRNA expression changes upon DNA damage-triggered p53 induction. In addition to miR-34a, a set of further miRNAs was found to be TP53 status dependently induced (particularly miR-182-5p, miR-7-5p and miR-320d/c). Beyond miRNAs, the present data demonstrate p53-dependent expression of the long non-coding RNAs lincRNA-p21 (long intergenic non-coding RNA p21) and NEAT1 (nuclear enriched abundant transcript 1) upon DNA damage and direct p53 activation with nutlin-3. p53-dependent induction of expression was further proven in a panel of Burkitt’s lymphoma (BL) cell lines including cell lines with genetically engineered knockout or knockdown of p53. p53 ChIP demonstrated direct binding of p53 to the NEAT1 promoter. This provides first evidence of p53-dependent regulation of long non-coding RNAs in CLL and BL. The discovery of p53-dependent NEAT1 induction, which is an integral part of nuclear paraspeckles, paves the way for further research on the role of paraspeckles in tumor cell apoptosis and resistence. The current work identifies additional components of the p53-dependent DNA damage response in lymphoma. The results of these studies provide new insight into the involvement of miRNAs and lncRNAs in two key signaling pathways regulating cell survival and treatment resistance in CLL and lymphoma

Investigating and reversing T-cell dysfunction in the Eμ-TCL1 mouse model of chronic lymphocytic leukaemia (CLL)

PhDChronic lymphocytic leukaemia (CLL) is the most common adult leukaemia, and despite recent introduction of targeted therapies, remains incurable. An important hallmark of CLL is severe immune deficiency, including the failure to mount effective anti-tumour immune responses. This can partly be explained by insufficient antigen presentation, but also by the existence of complex CLL-induced T-cell defects. Based on the cancer immuno-editing hypothesis that the immune system not only protects a host against tumour formation but can also be compromised to actively provide a pro-tumour microenvironment, modulating cancer-induced T-cell defects could restore the full anti-tumour response and result in more durable clinical responses. The immune checkpoint molecules PD-1 (expressed on activated immune effector cells) and PD-L1 (expressed on antigen-presenting and microenvironmental cells including tumour cells) have emerged as important mediators of T-cell suppression. Several studies suggest that PD-L1/PD-1 inhibitory signalling in CLL might be overcome by the immune modulatory drug lenalidomide. Furthermore, directly targeting PDL-1/PD-1 interactions produces significant responses in solid cancers. However, similar studies are notably absent in CLL, and the effect of PDL-1/PD-1 blockade on restoring cancer-induced immune dysfunction is not understood. Transgenic Eμ-TCL1 mice have been extensively validated as an adequate preclinical model of aggressive human CLL, and our group showed their suitability to mirror T-cell defects observed in human CLL. Using the Eμ-TCL1 model, this dissertation project substantially extends our previous characterization of CLL-induced T-cell dysfunction and evaluates the functional impact of PD-L1/PD-1 inhibitory signalling both in parallel with disease development and in different microenvironments. The findings to be described here demonstrate that developing CLL is associated with specific T-cell subset alterations, phenotypic changes, and functional defects that are very similar in peripheral blood and secondary lymphoid organs. In addition to PD-L1, PD-L2 is identified as a potential mediator of inhibitory signalling in CLL. CD8+ T cells in leukaemic mice are characterised as a functionally heterogeneous population, in which subsets of cells are able to exert effector functions despite PD-1 expression. In vivo lenalidomide treatment repairs selected phenotypic alterations and immune synapse formation, and a PD-L1 IgG blocking antibody effectively controls disease and reverses global T-cell defects even in cells expressing PD-1. In sum, this work provides a strong rationale to explore PD-L1/PD-1 targeting in CLL clinical trials, potentially in combination with novel agents.Mildred-Scheel Postdoctoral Fellowship awarded by Deutsche Krebshilfe (salary 2011-2013), by the Virtual Helmholtz Institute “VH404 - Resistance in Leukaemia” (salary and consumables, 2013-2015), and by the Kenneth Street Endowment Fund (consumables, 2011-2015)

The role of the NFAT signalling pathway on diffuse large B-cell lymphoma

PhD ThesisDiffuse Large B-Cell Lymphomas (DLBCL) are common, aggressive malignancies of mature B-lymphocytes that represent ~40% of lymphomas. Despite the widespread use of combined immunochemotherapy, approximately 50% of patients with DLBCL die from their disease. The two main DLBCL subgroups resemble activated B cells (ABC) or germinal centre B cells (GCB), where patients with ABC-DLBCL have significantly worse outcome. There is urgent need for novel therapeutic strategies in the treatment of DLBCL, which requires a better understanding of the molecular pathways upon which tumours depend. Accumulating evidence suggests that the signalling networks promoting and sustaining DLBCL derive from dysregulation of the normal pathways controlling B-lymphocyte activation and differentiation. There is increasing evidence indicating important roles for the NFAT family of transcription factors in DLBCL. Constitutively-active nuclear NFAT2 has been demonstrated in approximately 40% of primary DLBCL samples and NFAT has been shown to regulate a small number of genes associated with DLBCL growth/survival. This project investigated the role of NFAT in DLBCL. Nuclear localisation and activation of NFAT family members were characterised in a panel of DLBCL cell lines and chemical inhibition of calcineurin/NFAT, using Cyclosporin A (CsA), indicated dependency on the calcineurin/NFAT pathway for survival. Gene expression microarray analysis performed in DLBCL cell lines treated with CsA revealed potential NFAT target genes involved in the tumour microenvironment and anergy. These data revealed that the cytokine TNFα was downregulated by CsA in ABC, but not GCB cell lines. TNFα expression has recently been reported a significant prognostic factor for OS and PFS in DLBCL and evidence suggests dependency of some DLBCL on autocrine TNFα signalling for survival. Biologically active TNFα was produced by DLBCL cell lines, however inhibition of TNFα signalling using Infliximab and Etanercept had no effect on cell viability, suggesting that TNFα may be functionally important in DLBCL by other mechanisms.Cancer Research U

The importance of IκBNS and Ca²+ signaling in DLBCL development

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disorder of mature B-lymphocytes presenting with 40% of novel lymphoma cases worldwide the most common subtype of adult Non-Hodgkin lymphoma. DLBCL is characterised by a high degree of heterogeneity regarding clinical, pathologic and molecular genetic issues. Gene expression profiling revealed distinct molecular DLBCL subtypes resembling germinal centre B-cells (GCB DLBCL) and activated B-cells (ABC DLBCL). Despite advances in therapy, the 3-year progression free survival rates of GCB and ABC DLBCL upon immunochemotherapy are still at 74% and 40%, respectively. Consequently, there is an obvious need for identifying new molecular targets and biomarkers serving as starting points to develop new, more efficient therapeutic strategies especially of the adverse ABC DLBCL subtype. In the present study we were able to demonstrate that IκBNS is constitutively expressed in ABC DLBCL cell lines and human biopsies, whereas IκBNS expression is absent in GCB DLBCL. We noticed that two IκBNS isoforms were detectable, the described p35 and a second larger IκBNS isoform exhibiting an additional N-terminal unstructured portion. Silencing of IκBNS led to a reduced growth of ABC DLBCL cell lines, suggesting an oncogenic function of IκBNS. Promoter studies suggested that IκBNS expression is under the control of NF-κB and NFAT signalling. Interestingly, we detected constitutive activation of NFAT in DLBCL cell lines, which was necessary to drive the expression of IκBNS in ABC DLBCL. Calcineurin inhibitors, which decrease the activity of NFAT proteins, did not only impair the expression of IκBNS, but also induced cell death in multiple ABC DLBCL cell lines. The expression of the pro-survival cytokines IL-6/-10 was markedly reduced by calcineurin inhibition, which might at least partially explain their toxicity in ABC DLBCL cell lines. With the help of phosphoproteomics, we identified several new calcineurin substrates in ABC DLBCL, including CD79, suggesting a more central role of calcineurin in tumorigenesis. Taken together, these results provide first insights into the essential function of IκBNS in the development of ABC DLBCL. Furthermore, we suggest IκBNS as a promising biomarker to discriminate ABC from GCB DLBCL. Finally, we propose that calcineurin inhibitors could have therapeutic potential for the treatment of aggressive ABC DLBCL