Maurice Reimann

Synthetic lethal metabolic targeting of cellular senescence in cancer therapy

Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3). Although therapy-induced senescence (TIS) improves long-term outcomes, potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eµ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h1 to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h1– lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously. SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12- and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting.

Tumor Stroma-Derived TGF-β Limits Myc-Driven Lymphomagenesis via Suv39h1-Dependent Senescence

Activated RAS/BRAF oncogenes induce cellular senescence as a tumor-suppressive barrier in early cancer development, at least in part, via an oncogene-evoked DNA damage response (DDR). In contrast, Myc activation-although producing a DDR as well-is known to primarily elicit an apoptotic countermeasure. Using the Emu-myc transgenic mouse lymphoma model, we show here in vivo that apoptotic lymphoma cells activate macrophages to secrete transforming growth factor beta (TGF-beta) as a critical non-cell-autonomous inducer of cellular senescence. Accordingly, neutralization of TGF-beta action, like genetic inactivation of the senescence-related histone methyltransferase Suv39h1, significantly accelerates Myc-driven tumor development via cancellation of cellular senescence. These findings, recapitulated in human aggressive B cell lymphomas, demonstrate that tumor-prompted stroma-derived signals may limit tumorigenesis by feedback senescence induction.

Opposing roles of NF-κB in anti-cancer treatment outcome unveiled by cross-species investigations

In malignancies, enhanced nuclear factor-κB (NF-κB) activity is largely viewed as an oncogenic property that also confers resistance to chemotherapy. Recently, NF-κB has been postulated to participate in a senescence-associated and possibly senescence-reinforcing cytokine response, thereby suggesting a tumor-restraining role for NF-κB. Using a mouse lymphoma model and analyzing transcriptome and clinical data from lymphoma patients, we show here that therapy-induced senescence presents with and depends on active NF-κB signaling, whereas NF-κB simultaneously promotes resistance to apoptosis. Further characterization and genetic engineering of primary mouse lymphomas according to distinct NF-κB-related oncogenic networks reminiscent of diffuse large B-cell lymphoma (DLBCL) subtypes guided us to identify Bcl2-overexpressing germinal center B-cell-like (GCB) DLBCL as a clinically relevant subgroup with significantly superior outcome when NF-κB is hyperactive. Our data illustrate the power of cross-species investigations to functionally test genetic mechanisms in transgenic mouse tumors that recapitulate distinct features of the corresponding human entity, and to ultimately use the mouse model-derived genetic information to redefine novel, clinically relevant patient subcohorts.

Combined inhibition of PI3K-related DNA damage response kinases and mTORC1 induces apoptosis in MYC-driven B-cell lymphomas.

Pharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.

Senescence-associated reprogramming promotes cancer stemness

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed ‘stemness’). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eμ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.

B-cell-specific conditional expression of Myd88(p.L252P) leads to the development of diffuse large B-cell lymphoma in mice

The adaptor protein MYD88 is critical for relaying activation of Toll-like receptor signaling to NF-κB activation. MYD88 mutations, particularly the p.L265P mutation, have been described in numerous distinct B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL). Twenty-nine percent of activated B-cell-type DLBCL (ABC-DLBCL), which is characterized by constitutive activation of the NF-κB pathway, carry the p.L265P mutation. In addition, ABC-DLBCL frequently displays focal copy number gains affecting BCL2 Here, we generated a novel mouse model in which Cre-mediated recombination, specifically in B cells, leads to the conditional expression of Myd88(p.L252P) (the orthologous position of the human MYD88(p.L265P) mutation) from the endogenous locus. These mice develop a lymphoproliferative disease and occasional transformation into clonal lymphomas. The clonal disease displays the morphologic and immunophenotypical characteristics of ABC-DLBCL. Lymphomagenesis can be accelerated by crossing in a further novel allele, which mediates conditional overexpression of BCL2 Cross-validation experiments in human DLBCL samples revealed that both MYD88 and CD79B mutations are substantially enriched in ABC-DLBCL compared with germinal center B-cell DLBCL. Furthermore, analyses of human DLBCL genome sequencing data confirmed that BCL2 amplifications frequently co-occurred with MYD88 mutations, further validating our approach. Finally, in silico experiments revealed that MYD88-mutant ABC-DLBCL cells in particular display an actionable addiction to BCL2. Altogether, we generated a novel autochthonous mouse model of ABC-DLBCL that could be used as a preclinical platform for the development and validation of novel therapeutic approaches for the treatment of ABC-DLBCL.

The Myc/macrophage tango: Oncogene-induced senescence, Myc style

Ras/Raf-prototypic oncogenes induce cellular senescence, a terminal cell-cycle arrest, as a default cellular safeguard program, while oncogenic Myc is known to rather promote apoptosis as the prime failsafe mechanism. We review and discuss here evidence for Myc-induced senescence - which is detectable to a limited degree as a cell-autonomous, direct response to Myc action, but occurs predominantly in a non-cell-autonomous fashion via crosstalk of the oncogene-driven cell population with non-neoplastic bystanders, namely cells of the host immune system, prompting them to release pro-senescent cytokines that strike back onto adjacent proliferating tumor cells. In particular, we discuss how Myc-evoked apoptosis serves as a signal for macrophage attraction and activation, followed by the secretion of TGF-β as a cytokine that is capable of terminally arresting Myc-driven lymphoma cells without causing further DNA damage and without launching a senescence-associated, pro-inflammatory, and, therefore, potentially detrimental cytokine response in the target population. In essence, non-cell-autonomous but still oncogene-orchestrated senescence is a functionally relevant, robustly tumor-suppressive principle with critical implications for conceptually novel anti-cancer therapies in the clinic.

Non-cell-autonomous tumor suppression: oncogene-provoked apoptosis promotes tumor cell senescence via stromal crosstalk

Activated oncogenes evoke cellular fail-safe programs such as apoptosis, senescence, or autophagy to protect the organism from the expansion of damaged and potentially harmful cells. Non-cell-autonomous interactions between tumor cells and nonmalignant bystander cells add to cell-autonomous modes of tumor suppression during tumor development and progression. In particular, the role of stroma or host immune cells converting tumor-generated signals into a response that feeds back to the tumor cell population has been experimentally underappreciated. Using the Eμ-myc transgenic mouse lymphoma model, we elucidated how constitutive Myc signaling indirectly promotes cellular senescence via cytokines that were released by nonmalignant cells in response to oncogene-evoked cell-autonomous effects. Specifically, Myc primarily promotes apoptosis in a subset of the tumor cell population, leading to the attraction of macrophages, which subsequently engulf the apoptotic remainders. Phagocytosis-activated macrophages, in turn, exhibit strongly increased secretion of various cytokines, among them transforming growth factor beta to an extent that is capable of inducing cellular senescence in surrounding malignant cells. Our findings, recapitulated in human aggressive B-cell lymphomas, unveil that apoptosis and senescence are not simply two context-dependent cell-autonomous choices of stress responses, but rather cooperate via extracellular mediators—namely cells of the innate immune system—to profoundly limit tumorigenesis in vivo. A deeper mechanistic understanding of the organismic interconnection between different fail-safe programs will help to identify cellular components of the tumor stroma and their signal mediators that are readily available to impose a second line of host defense against cancer cells. This will open new perspectives for the development of antineoplastic therapies, whose targets not only encompass tumor but also stroma cell populations.

Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma

Classical Hodgkin lymphoma (cHL), although originating from B cells, is characterized by the virtual lack of gene products whose expression constitutes the B-cell phenotype. Epigenetic repression of B-cell-specific genes via promoter hypermethylation and histone deacetylation as well as compromised expression of B-cell-committed transcription factors were previously reported to contribute to the lost B-cell phenotype in cHL. Restoring the B-cell phenotype may not only correct a central malignant property, but it may also render cHL susceptible to clinically established antibody therapies targeting B-cell surface receptors or small compounds interfering with B-cell receptor signaling. We conducted a high-throughput pharmacological screening based on >28 000 compounds in cHL cell lines carrying a CD19 reporter to identify drugs that promote reexpression of the B-cell phenotype. Three chemicals were retrieved that robustly enhanced CD19 transcription. Subsequent chromatin immunoprecipitation-based analyses indicated that action of 2 of these compounds was associated with lowered levels of the transcriptionally repressive lysine 9-trimethylated histone H3 mark at the CD19 promoter. Moreover, the antileukemia agents all-trans retinoic acid and arsenic trioxide (ATO) were found to reconstitute the silenced B-cell transcriptional program and reduce viability of cHL cell lines. When applied in combination with a screening-identified chemical, ATO evoked reexpression of the CD20 antigen, which could be further therapeutically exploited by enabling CD20 antibody-mediated apoptosis of cHL cells. Furthermore, restoration of the B-cell phenotype also rendered cHL cells susceptible to the B-cell non-Hodgkin lymphoma-tailored small-compound inhibitors ibrutinib and idelalisib. In essence, we report here a conceptually novel, redifferentiation-based treatment strategy for cHL.

Erratum: Targeting the Senescence-Overriding Cooperative Activity of Structurally Unrelated H3K9 Demethylases in Melanoma (Cancer Cell (2018) 33(2) (322–336.e8) (S1535610818300023) (10.1016/j.ccell.2018.01.002))

Yong Yu, Kolja Schleich, Bin Yue, Sujuan Ji, Philipp Lohneis, Kristel Kemper, Mark R. Silvis, Nouar Qutob, Ellen van Rooijen, Melanie Werner-Klein, Lianjie Li, Dhriti Dhawan, Svenja Meierjohann, Maurice Reimann, Abdel Elkahloun, Steffi Treitschke, Bernd Dörken, Christian Speck, Frédérick A. Mallette, Leonard I. Zon, Sheri L. Holmen, Daniel S. Peeper, Yardena Samuels, Clemens A. Schmitt,* and Soyoung Lee *Correspondence: clemens.schmitt@charite.de https://doi.org/10.1016/j.ccell.2018.03.009