Lukas W. Pfannenstiel

Inhibition of Mcl-1 Promotes Senescence in Cancer Cells: Implications for Preventing Tumor Growth and Chemotherapy Resistance

ABSTRACT Although senescence in oncogenesis has been widely studied, little is known regarding the role of this process in chemotherapy resistance. Thus, from the standpoint of enhancing and improving cancer therapy, a better understanding of the molecular machinery involved in chemotherapy-related senescence is paramount. We show for the first time that Mcl-1, a Bcl-2 family member, plays an important role in preventing chemotherapy-induced senescence (CIS). Overexpression of Mcl-1 in p53+ cell lines inhibits CIS. Conversely, downregulation of Mcl-1 makes cells sensitive to CIS. Surprisingly, downregulation of Mcl-1 in p53− cells restored CIS to similar levels as p53+ cells. In all cases where senescence can be induced, we observed increased p21 expression. Moreover, we show that the domain of Mcl-1 responsible for its antisenescent effects is distinct from that known to confer its antiapoptotic qualities. In vivo we observe that downregulation of Mcl-1 can almost retard tumor growth regardless of p53 status, while overexpression of Mcl-1 in p53+ cells conferred resistance to CIS and promoted tumor outgrowth. In summary, our data reveal that Mcl-1 can inhibit CIS in both a p53-dependent and -independent manner in vitro and in vivo and that this Mcl-1-mediated inhibition can enhance tumor growth in vivo.

Interleukin-7 inhibits tumor-induced CD27-CD28- suppressor T cells: implications for cancer immunotherapy.

Purpose: We have previously reported that many types of tumors can induce changes in human T cells that lead to the acquisition of suppressive function and phenotypic alterations resembling those found in senescent T cells. In the present study, we find a role for interleukin 7 (IL-7) in protecting T cells from these changes and further define involved signaling pathways. Experimental Design: We evaluated the ability of IL-7 treatment to prevent the gain of suppressive function and phenotypic alterations in human T cells after a short coculture with tumor cells in vitro. We then used inhibitors of components of the phosphoinositide 3-kinase (PI3K)/AKT pathway and short interfering RNA knockdown of Mcl-1 and Bim to evaluate the role of these signaling pathways in IL-7 protection. Results: We found that IL-7 inhibits CD27/CD28 loss and maintains proliferative capacity, IL-2 production, and reduced suppressive function. The protective ability of IL-7 depended on activation of the PI3K/AKT pathway, which inhibited activation of glycogen synthase kinase 3β, which, in turn, prevented the phosphorylation and loss of Mcl-1. We further showed a key role for Mcl-1 in that its knockdown or inhibition abrogated the effects of IL-7. In addition, knockdown of the Mcl-1 binding partner and proapoptotic protein Bim protected T cells from these dysfunctional alterations. Conclusion: These observations confirm the role for Bcl-2 family members in cytokine signaling and suggest that IL-7 treatment in combination with other immunotherapies could lead to new clinical strategies to maintain normal T-cell function and reduce tumor-induced generation of dysfunctional and suppressor T cells. Clin Cancer Res; 17(15); 4975–86. ©2011 AACR.

Mcl-1 regulates reactive oxygen species via NOX4 during chemotherapy-induced senescence

Mcl-1, a Bcl-2 family member, is highly expressed in a variety of human cancers and is believed to enhance tumorigenic potential and chemotherapy resistance through the inhibition of apoptosis and senescence. We previously reported that Mcl-1′s regulation of chemotherapy-induced senescence (CIS) is dependent on its ability to prevent reactive oxygen species (ROS) generation. In this report, we demonstrate that Mcl-1-regulated CIS requires not only ROS, but specifically mitochondrial ROS, and that these events are upstream of activation of the DNA damage response, another necessary step toward senescence. Mcl-1′s anti-senescence activity also involves the unique ability to inhibit ROS formation by preventing the upregulation of pro-oxidants. Specifically, we found that NADPH oxidases (NOXs) are regulated by Mcl-1 and that NOX4 expression in particular is a required step for CIS induction that is blocked by Mcl-1. Lastly, we illustrate that by preventing expression of NOX4, Mcl-1 limits its availability in the mitochondria, thereby lowering the production of mitochondrial ROS during CIS. Our studies not only define the essential role of Mcl-1 in chemoresistance, but also for the first time link a key pro-survival Bcl-2 family member with the NOX protein family, both of which have significant ramifications in cancer progression.

Tumor-derived exosomes induce CD8+T cell suppressors

Background The suppressive nature of immune cells in the tumor microenvironment plays a major role in regulating anti-tumor immune responses. Our previous work demonstrated that a soluble factor from tumor cells is able to induce a suppressor phenotype (SP) in human CD8+ T cells typified by loss of CD27/CD28 expression and acquisition of a potent suppressor function. The present study hypothesized that the soluble mechanism that is inducing the SP in CD8+ T cells are tumor-derived exosomes (TDEs). Methods Membrane vesicles and TDEs from multiple head and neck cancer cell line’s conditioned growth media were isolated by ultracentrifugation and precipitation, respectively. Human purified CD3+CD8+ T cells were assessed for their induction of the T cell SP by flow cytometry identifying loss of CD27/CD28 expression and in vitro suppression assays. Furthermore, the T cell SP was characterized for the attenuation of IFN-γ production. To delineate exosomal proteins contributing to T cell SP, mass spectrometry was used to identify unique proteins that were present in TDEs. CRISPR/Cas9 knockout constructs were used to examine the role of one of these proteins, galectin-1. To assess the role of exosomal RNA, RNA purified from TDEs was nucleofected into CD8+ T cells followed by suppression analysis. Results Using fractionated conditioned growth media, factors >200 kDa induced CD8+ T cell SP, which was determined to be an exosome by mass spectrometry analysis. Multiple head and neck cancer-derived cell lines were found to secrete T cell SP-inducing exosomes. Mass spectrometry analysis revealed that an immunoregulatory protein, galectin-1 (Gal-1), was expressed in those exosomes, but not in TDEs unable to induce T cell SP. Galectin-1 knockout cells were found to be less able to induce T cell SP. Furthermore, RNA purified from the T cell SP-inducing exosomes were found to partially induce the SP when transfected into normal CD8+ T cells. Conclusions For the first-time, TDEs have been identified to induce a SP in CD8+ T cells and their mode of action may be synergistic effects from exosomal proteins and RNA. One protein in particular, galectin-1, appears to play a significant role in inducing T cell SP. Therefore, tumor-derived immunosuppressive exosomes are a potential therapeutic target to prevent T cell dysfunction and enhance anti-tumor immune responses.

Structure-Function Analysis of the Mcl-1 Protein Identifies a Novel Senescence-regulating Domain

Background: Mcl-1 is a prosurvival gene critical for chemotherapy resistance. However, its antisenescence properties are poorly characterized. Results: Through mutagenesis of Mcl-1 in functional assays, we identified a loop domain required for inhibiting senescence. Conclusion: An internal loop domain of Mcl-1 is responsible for antisenescence functions. Significance: Our study provides additional targets within Mcl-1 that can enhance senescence-inducing cancer treatments. Unlike other antiapoptotic Bcl-2 family members, Mcl-1 also mediates resistance to cancer therapy by uniquely inhibiting chemotherapy-induced senescence (CIS). In general, Bcl-2 family members regulate apoptosis at the level of the mitochondria through a common prosurvival binding groove. Through mutagenesis, we determined that Mcl-1 can inhibit CIS even in the absence of its apoptotically important mitochondrion-localizing domains. This finding prompted us to generate a series of Mcl-1 deletion mutants from both the N and C termini of the protein, including one that contained a deletion of all of the Bcl-2 homology domains, none of which impacted anti-CIS capabilities. Through subsequent structure-function analyses of Mcl-1, we identified a previously uncharacterized loop domain responsible for the anti-CIS activity of Mcl-1. The importance of the loop domain was confirmed in multiple tumor types, two in vivo models of senescence, and by demonstrating that a peptide mimetic of the loop domain can effectively inhibit the anti-CIS function of Mcl-1. The results from our studies appear to be highly translatable because we discerned an inverse relationship between the expression of Mcl-1 and of various senescence markers in cancerous human tissues. In summary, our findings regarding the unique structural properties of Mcl-1 provide new approaches for targeted cancer therapy.

Raiding the pharmacy: genomic screening identifies known chemotherapies as negative regulators of MCL1

Despite multiple studies demonstrating the importance of the anti-apoptotic proteinMcl-1 in tumor cell survival and treatment resistance, a clinically importantinhibitor has yet to be developed. A recent study by Guo Wei and colleagues publishedin Cancer Cell has utilized a novel high-throughput approach to identifycompounds that act as transcriptional repressors of MCL1 expression. Theirfindings identified a number of candidate drugs to be tested for clinical relevancein human cancers dependent on MCL1 expression.

Harnessing the potential of radiation treatment of brain metastases to improve melanoma immunotherapy

Radiation is an integral part of melanoma therapy, particularly for patients with symptomatic metastases. Efficient killing of tumor cells by radiation has been shown in immunocompromised mouse models to be enhanced greatly with transfer of a functioning immune system, particularly CD8+ T cells. In a subset of patients, addition of radiation to immunotherapy has also enhanced immune system-mediated tumor cell kill in non-irradiated areas, termed abscopal effect. This effect is thought to be a result of increased shedding of tumor antigens upon radiotherapy that primes the immune system to establish a more robust attack on melanoma cells throughout the body. Whether or not brain metastases in the blood brain barrier can be used to develop an abscopal effect is unknown. Pre-clinical reports support synergism between PD-1 inhibition and radiation in tumor killing and prolonged survival.

Mcl-1 expression influcences CD8+ anti-tumor immunity.

Meeting abstracts The recent clinical successes of immunotherapies such as anti-PD-1 and anti-CTLA-4 antibody blockade have re-invigorated the idea that the immune system has the potential to be a powerful tool to destroy tumors. However despite ample evidence that these therapies are able to

Abstract P33: A Distinct Domain of Mcl1 Regulates Senescence Inhibition in Cancer

Abeba Demelash, PhD1; Lukas W Pfannenstiel, PhD2; Brian T Maybruck, PhD2; Simon E Schlanger, BS2; Tannenbaum S Charles, PhD2; Brian R Gastman, MD2 1Institutes of Head and Neck, Dermatology and Plastic Surgery, Taussing Cancer Center and Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, Cleveland, OH, Institutes of Head and Neck, Dermatology and Plastic Surgery, Taussing Cancer Center and Lerner Research Institute, Cleveland, OH

Abstract P34: Tumor-Induced Suppressive CD8+ T Cells

Abeba Demelash, PhD1; Lukas W Pfannenstiel, PhD2; Brian T Maybruck, PhD2; Simon E Schlanger, BS2; Tannenbaum S Charles, PhD2; Brian R Gastman, MD2 1Institutes of Head and Neck, Dermatology and Plastic Surgery, Taussing Cancer Center and Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, Cleveland, OH, Institutes of Head and Neck, Dermatology and Plastic Surgery, Taussing Cancer Center and Lerner Research Institute, Cleveland, OH