Consensus guidelines for the definition, detection and interpretation of immunogenic cell death.

Cells succumbing to stress via regulated cell death (RCD) can initiate an adaptive immune response associated with immunological memory, provided they display sufficient antigenicity and adjuvanticity. Moreover, multiple intracellular and microenvironmental features determine the propensity of RCD to drive adaptive immunity. Here, we provide an updated operational definition of immunogenic cell death (ICD), discuss the key factors that dictate the ability of dying cells to drive an adaptive immune response, summarize experimental assays that are currently available for the assessment of ICD in vitro and in vivo, and formulate guidelines for their interpretation.

Dinaciclib induces immunogenic cell death and enhances anti-PD1-mediated tumor suppression.

Blockade of the checkpoint inhibitor programmed death 1 (PD1) has demonstrated remarkable success in the clinic for the treatment of cancer; however, a majority of tumors are resistant to anti-PD1 monotherapy. Numerous ongoing clinical combination therapy studies will likely reveal additional therapeutics that complement anti-PD1 blockade. Recent studies found that immunogenic cell death (ICD) improves T cell responses against different tumors, thus indicating that ICD may further augment antitumor immunity elicited by anti-PD1. Here, we observed antitumor activity following combinatorial therapy with anti-PD1 Ab and the cyclin-dependent kinase inhibitor dinaciclib in immunocompetent mouse tumor models. Dinaciclib induced a type I IFN gene signature within the tumor, leading us to hypothesize that dinaciclib potentiates the effects of anti-PD1 by eliciting ICD. Indeed, tumor cells treated with dinaciclib showed the hallmarks of ICD including surface calreticulin expression and release of high mobility group box 1 (HMGB1) and ATP. Mice treated with both anti-PD1 and dinaciclib showed increased T cell infiltration and DC activation within the tumor, indicating that this combination improves the overall quality of the immune response generated. These findings identify a potential mechanism for the observed benefit of combining dinaciclib and anti-PD1, in which dinaciclib induces ICD, thereby converting the tumor cell into an endogenous vaccine and boosting the effects of anti-PD1.

Serum-resistant CpG-STAT3 decoy for targeting survival and immune checkpoint signaling in acute myeloid leukemia.

Targeting oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) in acute myeloid leukemia (AML) can reduce blast survival and tumor immune evasion. Decoy oligodeoxynucleotides (dODNs), which comprise STAT3-specific DNA sequences are competitive inhibition of STAT3 transcriptional activity. To deliver STAT3dODN specifically to myeloid cells, we linked STAT3dODN to the Toll-like receptor 9 (TLR9) ligand, cytosine guanine dinucleotide (CpG). The CpG-STAT3dODN conjugates are quickly internalized by human and mouse TLR9(+)immune cells (dendritic cells, B cells) and the majority of patients' derived AML blasts, including leukemia stem/progenitor cells. Following uptake, CpG-STAT3dODNs are released from endosomes, and bind and sequester cytoplasmic STAT3, thereby inhibiting downstream gene expression in target cells. STAT3 inhibition in patients' AML cells limits their immunosuppressive potential by reduced arginase expression, thereby partly restoring T-cell proliferation. Partly chemically modified CpG-STAT3dODNs have >60 hours serum half-life which allows for IV administration to leukemia-bearing mice (50% effective dose ∼ 2.5 mg/kg). Repeated administration of CpG-STAT3dODN resulted in regression of human MV4-11 AML in mice. The antitumor efficacy of this strategy is further enhanced in immunocompetent mice by combining direct leukemia-specific cytotoxicity with immunogenic effects of STAT3 blocking/TLR9 triggering. CpG-STAT3dODN effectively reducedCbfb/MYH11/MplAML burden in various organs and eliminated leukemia stem/progenitor cells, mainly through CD8/CD4 T-cell-mediated immune responses. In contrast, small-molecule Janus kinase 2/STAT3 inhibitor failed to reproduce therapeutic effects of cell-selective CpG-STAT3dODN strategy. These results demonstrate therapeutic potential of CpG-STAT3dODN inhibitors with broad implications for treatment of AML and potentially other hematologic malignancies.

TLR9-Targeted SiRNA Delivery In Vivo.

The SiRNA strategy is a potent and versatile method for modulating expression of any gene in various species for investigational or therapeutic purposes. Clinical translation of SiRNA-based approaches proved challenging, mainly due to the difficulty of targeted SiRNA delivery into cells of interest and the immunogenic side effects of oligonucleotide reagents. However, the intrinsic sensitivity of immune cells to nucleic acids can be utilized for the delivery of SiRNAs designed for the purpose of cancer immunotherapy. We have demonstrated that synthetic ligands for the intracellular receptor TLR9 can serve as targeting moiety for cell-specific delivery of SiRNAs. Chemically synthesized CpG-SiRNA conjugates are quickly internalized by TLR9-positive cells in the absence of transfection reagents, inducing target gene silencing. The CpG-SiRNA strategy allows for effective targeting of TLR9-positive cells in vivo after local or systemic administration of these oligonucleotides into mice.

Pazopanib as third line therapy for metastatic renal cell carcinoma: clinical efficacy and temporal analysis of cytokine profile.

PURPOSE: Pazopanib has been assessed primarily in cytokine refractory or treatment naïve patients with metastatic renal cell carcinoma. Outcomes have been associated with a specific immunological profile. However, pazopanib activity in the third line setting and temporal changes in the immunological profile during therapy are poorly understood. MATERIALS AND METHODS: Study eligibility was limited to patients with 2 prior lines of therapy, including at least 1 vascular endothelial growth factor directed therapy, as well as ECOG performance status 0 to 2 and clear cell histology. Patients received pazopanib 800 mg daily. A Simon minmax 2-stage design was used with 80% power to determine an encouraging 23% overall response rate (10% type I error). Immunological profiles were assessed monthly on a Luminex® platform using the Human Cytokine 30-Plex Cytokine Immunoassay (Invitrogen™). RESULTS: A total of 28 patients with a median age of 63 years (range 45 to 86) were enrolled in study. Of the patients 12 (43%) had a confirmed complete (1) or partial (11) response. In the cohort median progression-free survival was 16.5 months (95% CI 14.7-not reached). The most common grade 3/4 toxicities were hypertension (46% of cases) and proteinuria (14%). At 6 and 12 months responders had lower levels of HGF, VEGF, IL-6 and 8, and soluble IL-2R (each p <0.05). Nonresponders also showed increased numbers of myeloid-derived suppressor cells at each interval. Phenotypic and functional studies confirmed that the myeloid-derived suppressor cells were granulocytic. CONCLUSIONS: Progression-free survival and the overall response rate associated with third line pazopanib were encouraging. Immunological profile differences between responders and nonresponders suggest that the mechanism of pazopanib resistance is at least partly related to the generation of systemic tumor immune tolerance.

Leukemia cell-targeted STAT3 silencing and TLR9 triggering generate systemic antitumor immunity.

Signal transducer and activator of transcription 3 (STAT3) is an oncogene and immune checkpoint commonly activated in cancer cells and in tumor-associated immune cells. We previously developed an immunostimulatory strategy based on targeted Stat3 silencing in Toll-like receptor 9 (TLR9)-positive hematopoietic cells using CpG-small interfering RNA (siRNA) conjugates. Here, we assessed the therapeutic effect of systemic STAT3 blocking/TLR9 triggering in disseminated acute myeloid leukemia (AML). We used mouse Cbfb-MYH11/Mpl-induced leukemia model, which mimics human inv(16) AML. Our results demonstrate that intravenously delivered CpG-Stat3 siRNA, but not control oligonucleotides, can eradicate established AML and impair leukemia-initiating potential. These antitumor effects require host's effector T cells but not TLR9-positive antigen-presenting cells. Instead, CpG-Stat3 siRNA has direct immunogenic effect on AML cells in vivo upregulating major histocompatibility complex class-II, costimulatory and proinflammatory mediators, such as interleukin-12, while downregulating coinhibitory PD-L1 molecule. Systemic injections of CpG-Stat3 siRNA generate potent tumor antigen-specific immune responses, increase the ratio of tumor-infiltrating CD8(+) T cells to regulatory T cells in various organs, and result in CD8(+) T-cell-dependent regression of leukemia. Our findings underscore the potential of using targeted STAT3 inhibition/TLR9 triggering to break tumor tolerance and induce immunity against AML and potentially other TLR9-positive blood cancers.

Retracted: FoxP3 acts as a cotranscription factor with STAT3 in tumor-induced regulatory T cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the authors. This study provided an explanation for why loss of FoxP3 in inducible regulatory T cells results in reduced expression of interleukin (IL)-10 despite the absence of FoxP3 binding sites in the IL-10 promoter. STAT3 binding sites do exist in the promoter, and evidence for a direct molecular interaction between FoxP3 and STAT3 proteins was provided as an explanation of the effect of loss of FoxP3. As supporting evidence, we reported modeling of a structural interaction between these two transcription factors in Figure 4D. As the N-terminal region of FoxP3, which consists of the Exon-2 region, had not been solved at structural resolution, we mistakenly used what we deduced to be a FoxP3 related transcription factor, NFAT, in the modeling. The model depicted in Figure 4D therefore did not represent a putative interaction between FoxP3 and STAT3 as labeled, but rather a putative interaction between NFAT and STAT3. Given the incorrect labeling of Figure 4D, the lack of documentation in the paper describing exactly how the modeling was performed, the lack of evidence shown in the paper for the choice of NFAT as the modeling partner, and the limited supporting evidence for a cooperative interaction between FoxP3 and STAT3, the editors have concluded with the corresponding author that the appropriate course of action is to retract the paper. We apologize for any confusion and inconvenience caused to readers.

Calcarea carbonica induces apoptosis in cancer cells in p53-dependent manner via an immuno-modulatory circuit.

BACKGROUND: Complementary medicines, including homeopathy, are used by many patients with cancer, usually alongside with conventional treatment. However, the molecular mechanisms underneath the anti-cancer effect, if any, of these medicines have still remained unexplored. To this end we attempted to evaluate the efficacy of calcarea carbonica, a homeopathic medicine, as an anti-cancer agent and to delineate the detail molecular mechanism(s) underlying calcerea carbonica-induced tumor regression. METHODS: To investigate and delineate the underlying mechanisms of calcarea carbonica-induced tumor regression, Trypan blue dye-exclusion test, flow cytometric, Western blot and reverse transcriptase-PCR techniques were employed. Further, siRNA transfections and inhibitor studies were used to validate the involvement of p53 pathway in calcarea carbonica-induced apoptosis in cancer cells. RESULTS: Interestingly, although calcarea carbonica administration to Ehrlich's ascites carcinoma (EAC)- and Sarcoma-180 (S-180)-bearing Swiss albino mice resulted in 30-35% tumor cell apoptosis, it failed to induce any significant cell death in ex vivo conditions. These results prompted us to examine whether calcarea carbonica employs the immuno-modulatory circuit in asserting its anti-tumor effects. Calcarea carbonica prevented tumor-induced loss of effector T cell repertoire, reversed type-2 cytokine bias and attenuated tumor-induced inhibition of T cell proliferation in tumor-bearing host. To confirm the role of immune system in calcarea carbonica-induced cancer cell death, a battery of cancer cells were co-cultured with calcarea carbonica-primed T cells. Our results indicated a "two-step" mechanism of the induction of apoptosis in tumor cells by calcarea carbonica i.e., (1) activation of the immune system of the host; and (2) induction of cancer cell apoptosis via immuno-modulatory circuit in p53-dependent manner by down-regulating Bcl-2:Bax ratio. Bax up-regulation resulted in mitochondrial transmembrane potential loss and cytochrome c release followed by activation of caspase cascade. Knocking out of p53 by RNA-interference inhibited calcarea carbonica-induced apoptosis thereby confirming the contribution of p53. CONCLUSION: These observations delineate the significance of immuno-modulatory circuit during calcarea carbonica-mediated tumor apoptosis. The molecular mechanism identified may serve as a platform for involving calcarea carbonica into immunotherapeutic strategies for effective tumor regression.

TLR9-mediated siRNA delivery for targeting of normal and malignant human hematopoietic cells in vivo.

STAT3 operates in both cancer cells and tumor-associated immune cells to promote cancer progression. As a transcription factor, it is a highly desirable but difficult target for pharmacologic inhibition. We have recently shown that the TLR9 agonists CpG oligonucleotides can be used for targeted siRNA delivery to mouse immune cells. In the present study, we demonstrate that a similar strategy allows for targeted gene silencing in both normal and malignant human TLR9(+) hematopoietic cells in vivo. We have developed new human cell-specific CpG(A)-STAT3 siRNA conjugates capable of inducing TLR9-dependent gene silencing and activation of primary immune cells such as myeloid dendritic cells, plasmacytoid dendritic cells, and B cells in vitro. TLR9 is also expressed by several human hematologic malignancies, including B-cell lymphoma, multiple myeloma, and acute myeloid leukemia. We further demonstrate that oncogenic proteins such as STAT3 or BCL-X(L) are effectively knocked down by specific CpG(A)-siRNAs in TLR9(+) hematologic tumor cells in vivo. Targeting survival signaling using CpG(A)-siRNAs inhibits the growth of several xenotransplanted multiple myeloma and acute myeloid leukemia tumors. CpG(A)-STAT3 siRNA is immunostimulatory and nontoxic for normal human leukocytes in vitro. The results of the present study show the potential of using tumoricidal/immunostimulatory CpG-siRNA oligonucleotides as a novel 2-pronged therapeutic strategy for hematologic malignancies.

Curcumin: the multi-targeted therapy for cancer regression.

Tumors are multifaceted; in fact, numerous things happen in synchrony to enable tumor promotion and progression. Any type of cancer is associated with the modification of 300-500 normal genes and characterized by the deregulation of cell signaling pathways at multiple steps leading to cancer phenotype. Thus a proper management of tumorigenesis requires the development of multi-targeted therapies. Several adverse effects associated with present day cancer therapies and the thirsts for multi-targeted safe anticancer drug instigate the use of natural polyphenol, curcumin. It appears to involve a blend of anti-carcinogenic, pro-apoptotic, anti-angiogenic, anti-metastatic, immunomodulatory and antioxidant activities. Also the molecular mechanisms implicated for the pleotropic activities of curcumin are diverse and seem to involve a combination of cell signaling pathways at multiple levels of tumorigenesis. Being a potent scavenger of reactive oxygen species, curcumin also ameliorates systemic toxicity in tumor-bearer. Taken together, by placing particular emphasis on molecular basis of tumor promotion and progression this review summarizes the anti-cancer properties of curcumin that may be exploited for successful clinical cancer prevention.

Tumor gangliosides and T cells: a deadly encounter.

Despite major advances in understanding the mechanisms of tumor immunity, its successful translation into effective tumor immunotherapy is hindered by the ability of tumors to foster a tolerant microenvironment and to activate a plethora of immunosuppressive mechanisms. Among different strategies employed by tumors to thwart immune responses, shedding of immunosuppressive molecules, such as sialic acid-containing glycosphingolipids, gangliosides, by the tumor is one important strategy. Aberrant and elevated expression of gangliosides has been demonstrated on the surface of cancer cells. Here we discuss about the molecular mechanisms underneath the contribution of tumor gangliosides in targeting multiple steps of T cell response. We shall also underscore the contribution of T-regulatory, NK and dendritic cells in this immunosuppressive network. Inhibitory effects of gangliosides ultimately converge to T cell apoptosis in receptor-dependent and -independent manners via IL-2 deprivation, ROS production, cytochrome c release, NFkappaB inhibition and caspase activation. Current wealth of information promises a future scenario in which synchronized blockade of immunosuppressive mechanisms and removal of inhibitory signals might be effective in overcoming immunological tolerance and promoting tumor regression.

Curcumin enhances the efficacy of chemotherapy by tailoring p65NFκB-p300 cross-talk in favor of p53-p300 in breast cancer.

Breast cancer cells often develop multiple mechanisms of drug resistance during tumor progression, which is the major reason for the failure of breast cancer therapy. High constitutive activation of NFκB has been found in different cancers, creating an environment conducive for chemotherapeutic resistance. Here we report that doxorubicin-induced SMAR1-dependent transcriptional repression and SMAR1-independent degradation of IkBα resulted in nuclear translocation of p65NFκB and its association with p300 histone acetylase and subsequent transcription of Bcl-2 to impart protective response in drug-resistant cells. Consistently SMAR1-silenced drug-resistant cells exhibited IkBα-mediated inhibition of p65NFκB and induction of p53-dependent apoptosis. Interestingly, curcumin pretreatment of drug-resistant cells alleviated SMAR1-mediated p65NFκB activation and hence restored doxorubicin sensitivity. Under such anti-survival condition, induction of p53-p300 cross-talk enhanced the transcriptional activity of p53 and intrinsic death cascade. Importantly, promyelocyte leukemia-mediated SMAR1 sequestration that relieved the repression of apoptosis-inducing genes was indispensable for such chemo-sensitizing ability of curcumin. A simultaneous decrease in drug-induced systemic toxicity by curcumin might also have enhanced the efficacy of doxorubicin by improving the intrinsic defense machineries of the tumor-bearer. Overall, the findings of this preclinical study clearly demonstrate the effectiveness of curcumin to combat doxorubicin-resistance. We, therefore, suggest curcumin as a potent chemo-sensitizer to improve the therapeutic index of this widely used anti-cancer drug. Taken together, these results suggest that curcumin can be developed into an adjuvant chemotherapeutic drug.

Gain of cellular adaptation due to prolonged p53 impairment leads to functional switchover from p53 to p73 during DNA damage in acute myeloid leukemia cells.

Tumor suppressor p53 plays the central role in regulating apoptosis in response to genotoxic stress. From an evolutionary perspective, the activity of p53 has to be backed up by other protein(s) in case of any functional impairment of this protein, to trigger DNA damage-induced apoptosis in cancer cells. We adopted multiple experimental approaches to demonstrate that in p53-impaired cancer cells, DNA damage caused accumulation of p53 paralogue p73 via Chk-1 that strongly impacted Bax expression and p53-independent apoptosis. On the contrary, when p53 function was restored by ectopic expression, Chk-2 induced p53 accumulation that in turn overshadowed p73 activity, suggesting an antagonistic interaction between p53 family members. To understand such interaction better, p53-expressing cells were impaired differentially for p53 activity. In wild-type p53-expressing cancer cells that were silenced for p53 for several generations, p73 was activated, whereas no such trend was observed when p53 was transiently silenced. Prolonged p53 interference, even in functional p53 settings, therefore, leads to the "gain of cellular adaptation" in a way that alters the cellular microenvironment in favor of p73 activation by altering p73-regulatory proteins, e.g. Chk1 activation and dominant negative p73 down-regulation. These findings not only unveil a hitherto unexplained mechanism underlying the functional switchover from p53 to p73, but also validate p73 as a promising and potential target for cancer therapy in the absence of functional p53.

Theaflavins retard human breast cancer cell migration by inhibiting NF-kappaB via p53-ROS cross-talk.

The present study demonstrates that theaflavins exploit p53 to impede metastasis in human breast cancer cells. Our data suggest that p53-dependent reactive oxygen species (ROS) induce p53-phosphorylation via p38MAPK in a feedback loop to inhibit IkappaBalpha-phosphorylation and NF-kappaB/p65 nuclear translocation, thereby down-regulating the metastatic proteins metalloproteinase (MMP)-2 and MMP-9. When wild-type p53-expressing MCF-7 cells are transfected with p53 short-interfering RNA, or treated with a pharmacological inhibitor of ROS, theaflavins fail to inhibit NF-kappaB-mediated cell migration. On the other hand, NF-kappaB over-expression bestows MCF-7 cells with resistance to the anti-migratory effect of theaflavins. These results indicate that inhibition of NF-kappaB via p53-ROS crosstalk is a pre-requisite for theaflavins to accomplish the anti-migratory effect in breast cancer cells.

Theaflavins target Fas/caspase-8 and Akt/pBad pathways to induce apoptosis in p53-mutated human breast cancer cells.

The most common alterations found in breast cancer are inactivation or mutation of tumor suppressor gene p53. The present study revealed that theaflavins induced p53-mutated human breast cancer cell apoptosis. Pharmacological inhibition of caspase-8 or expression of dominant-negative (Dn)-caspase-8/Fas-associated death domain (FADD) partially inhibited apoptosis, whereas caspase-9 inhibitor completely blocked the killing indicating involvement of parallel pathways that converged to mitochondria. Further studies demonstrated theaflavin-induced Fas upregulation through the activation of c-jun N-terminal kinase, Fas-FADD interaction in a Fas ligand-independent manner, caspase-8 activation and t-Bid formation. A search for the parallel pathway revealed theaflavin-induced inhibition of survival pathway, mediated by Akt deactivation and Bcl-xL/Bcl-2-associated death promoter dephosphorylation. These well-defined routes of growth control converged to a common process of mitochondrial transmembrane potential loss, cytochrome c release and activation of the executioner caspase-9 and -3. Overexpression of either constitutively active myristylated-Akt (Myr-Akt) or Dn-caspase-8 partially blocked theaflavin-induced mitochondrial permeability transition and apoptosis of p53-mutated cells, whereas cotransfection of Myr-Akt and Dn-caspase-8 completely abolished theaflavin effect thereby negating the possibility of existence of third pathways. These results and other biochemical correlates established the concept that two distinct signaling pathways were regulated by theaflavins to induce mitochondrial death cascade, eventually culminating to apoptosis of p53-mutated human breast cancer cells that are strongly resistant to conventional therapies.