PARP inhibitors induce a senescence phenotype in non-small cell lung carcinoma cell lines.

TLR7 agonists are currently under investigations for their ability to enhance anti-tumor immune responses. However, in some tumor models, these agonists also stimulate tumor cells, which can express high levels of TLR7, a receptor for single-stranded RNA [1]. We have demonstrated that stimulation of lung tumor cell lines expressing TLR7 with synthetic TLR7 agonists led to upregulation of the antiapoptotic protein Bcl-2, tumor cell survival and chemoresistance [2]. In Non-Small-Cell Lung Carcinoma (NSCLC) cohorts of patients, we have observed high expression of TLR7 on tumor cells of 70% of patients, which conferred poor clinical outcome and was strongly associated with resistance to chemotherapy [3]. This pro-tumoral effect of TLR7 has been validated in murine models in which the injection of TLR7 agonists in NOD/SCID mice, in C57BL/6 wild-type or in TLR7-deficient mice grafted with lung adenocarcinoma tumor cells led to increased tumor progression, increased lung metastasis, and resistance to chemotherapy. On the contrary, we demonstrated that TLR7 antagonist injection led to antitumoral effect. Additionally, TLR7 stimulation resulted in a significant increase of Myeloid-Derived Suppressor Cells (MDSC) in the tumor microenvironment. Depletion experiments of MDSC indicated that these cells are involved in the pro-tumoral effect induced upon TLR7 stimulation. Finally, we have demonstrated that the pro-tumoral effect of TLR7 stimulation, mediated by the MDSC recruitment, was independent of TLR7 stimulation on immune cells. Our results reveal the mechanism by which TLR7 stimulation induce the pro-tumoral effect in mice, and open the way to the development of novel cancer therapeutics including TLR7 inhibitors, for NSCLC patients. This results demonstrate the important role of TLR7 in lung tumor progression. Knowing that natural ligands of TLR7 are ssRNA, we suppose that this effect could be linked to viral infections or RNA released in the tumor microenvironment. [1] Dajon, et al. Immunobiologie. 2016 [2] Cherfils-Vicini J, et al. J Clin Invest. 2010 [3] Chatterjee J, et al. Cancer Res. 2014 Citation Format: Marion Dajon, Kristina Iribarren, Marco Alifano, Diane Damotte, Isabelle Cremer. Protumoral and pro-metastatic effects of TLR7 in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5752.

PARP inhibitor and chemotherapy combination trials for the treatment of advanced malignancies: does a development pathway forward exist?

Significant advances in our understanding of neutrophil biology were made in the past several years. A newly discovered mechanism was discovered, the formation of neutrophils extracellular traps (NETs). The structure of NETs is composed of the DNA strand and neutrophil granule proteins. NETs were found to have an association with tumor progression. This review highlights the latest knowledge about the controversial effect on tumors of NETs. Pro-tumor and anti-tumor effects are described respectively. The probable mechanisms of the anti-tumor effect are related to its direct killing of cancer cells or stimulation of the immune system to fight against the tumor. The pro-tumor effect has a correlation with matrix metalloproteinase 9 (MMP-9), cathepsin G, and neutrophil elastase (NE). Moreover, the structure of the NETs makes it able to catch the circulating tumor cells, which could lead to metastasis. This review summarizes our knowledge about the proven roles of NETs in the progression of cancer with particular focus on the components of the NETs, and considers NETs as a potential target for cancer therapy.

TLR7 agonists are currently under investigations for their ability to enhance anti-tumor immune responses. However, in some tumor models, these agonists can also stimulate tumor cells, which express high levels of TLR7. In Non-Small-Cell Lung Carcinoma (NSCLC), we have demonstrated that high expression of TLR7 conferred poor clinical outcome and was strongly associated with resistance to chemotherapy. This protumorigenic effect of TLR7 has been validated in murine models in which the injection of TLR7 agonists in NOD/SCID mice, in C57BL/6 wild-type or in TLR7-deficient mice grafted with lung adenocarcinoma tumor cells led to increased tumor progression and chemotherapeutic resistance. TLR7 stimulation resulted in a significant increase of Myeloid-Derived Suppressor Cells (MDSC) in the tumor microenvironment. Depletion experiments of MDSC indicated that these cells appeared to be responsible to the in vivo pro-tumoral effect induced upon TLR7 stimulation. Finally, we have demonstrated that the pro-tumoral effect of TLR7 stimulation, mediated by the MDSC recruitment, was independent of stimulation of TLR7 of immune cells. Our results reveal the mechanism by which TLR7 stimulation induce the pro-tumoral effect in mice, and open the way to the development of novel cancer therapeutics including TLR7 inhibitors, for NSCLC patients. Citation Format: Marion Dajon, Kristina Iribarren, Isabelle Cremer. Protumoral effects of TLR7 in lung tumors [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A124.

Simple SummaryRadiotherapy is one of the most common treatments for cancer. Overcoming the failure and side effects of radiotherapy are current challenges. It has been recently demonstrated that senescence contributes to radioresistance. Cellular senescence is a permanent arrest in cell proliferation induced by various factors, such as radiation. Here, we aimed to assess the potential of combining the radiation and DNA damage repair inhibitor, Olaparib to a senolytic drug, ABT-263. We demonstrated that combining radiation, Olaparib and ABT-263 successfully targeted the radio-induced senescent cells resulting in increased cell death and reduced senescence-associated secretory phenotype. These results paved the way towards a new therapeutic combination for patients treated with radiotherapy and Olaparib.Radiotherapy (RT) is a key component of cancer treatment. Although improvements have been made over the years, radioresistance remains a challenge. For this reason, a better understanding of cell fates in response to RT could improve therapeutic options to enhance cell death and reduce adverse effects. Here, we showed that combining RT (photons and protons) to noncytotoxic concentration of PARP inhibitor, Olaparib, induced a cell line-dependent senescence-like phenotype. The senescent cells were characterized by morphological changes, an increase in p21 mRNA expression as well as an increase in senescence-associated β-galactosidase activity. We demonstrated that these senescent cells could be specifically targeted by Navitoclax (ABT-263), a Bcl-2 family inhibitor. This senolytic drug led to significant cell death when combined with RT and Olaparib, while limited cytotoxicity was observed when used alone. These results demonstrate that a combination of RT with PARP inhibition and senolytics could be a promising therapeutic approach for cancer patients.

Background: Inhibition of poly (ADP-ribose) polymerase (PARP) is an exciting treatment strategy recently approved for prostate cancer patients with homologous recombination repair defects. Despite this advance in the field, it remains unclear how PARP inhibitor (PARPi) sensitive cells respond to treatment. We previously demonstrated that treatment with the PARPi olaparib induces not only cell death, but also G2/M arrested senescence characterized by activation of the p53 signaling pathway. We hypothesize that targeting PARPi induced senescence may provide a means to enhance the efficacy of PARPi treatment. In our current work, we sought to 1) understand whether senescence induction is a generalized response to all PARPi’s and 2) characterize senescence induction to guide the development of novel treatment strategies combining a PARPi with a senolytic drug. Methods: PARPi sensitive LNCaP and C4-2B prostate tumor cells were treated with olaparib, rucaparib, niraparib, or talazoparib for 5 days to induce senescence. Both vehicle treated and quiescent cells (LNCaP and C4-2B cultured in FBS-low (0.2%) conditions) were used as controls. Cell viability, flow cytometry, and beta-galactosidase activity assays tested response to PARPi’s. Western blot was used to detect PARP activity, apoptosis, and DNA damage response. RNA-sequencing was performed to characterize senescence induced signaling alterations. Results: We found that exposure to rucaparib, niraparib, and talazoparib all induce a robust G2/M arrested senescence response in LNCaP and C4-2B cells, suggesting that senescence induction is a class effect of PARPi’s. PARPi induced senescence is characterized by activation of the p53 signaling pathway and significantly increased expression of the cyclin-dependent kinase inhibitor p21. Furthermore, PARPi induced senescence is distinct from quiescence, suggesting that response to PARP inhibition is phenotypically different from a more general growth arrest. RNA-sequencing revealed several signaling changes associated with senescence which may provide novel treatment opportunities. Conclusions: Senolytics are a class of drugs thought to specifically target senescent cells. Our results demonstrate that PARPi’s induce senescence. Future work will be directed at further characterizing PARPi induced senescence, leading to rationally selected senolytic drug combinations which may enhance the efficacy of PARPi therapy. Citation Format: Alan P. Lombard, Wei Lou, Christopher P. Evans, Allen C. Gao. Characterizing senescence response to PARP inhibition may provide opportunities for enhanced efficacy through combinations with senolytic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1850.

Recent studies suggest that PARP1 inhibitors, several of which are currently in clinical trial, may selectively kill BRCA1/2 mutant cancers cells. It is thought that the success of this therapy is based on immitigable lethal DNA damage in the cancer cells resultant from the concurrent loss or inhibition of two DNA damage repair pathways: single-strand break (SSB) repair and homologous recombination repair (HRR). Presumably, inhibition of PARP1 activity obstructs the repair of SSBs and during DNA replication, these lesions cause replication fork collapse and are transformed into substrates for HRR. In fact, several previous studies have indicated a hyper-recombinogenic phenotype in the absence of active PARP1 in vitro or in response to DNA damaging agents. In this study, we demonstrate an increased frequency of spontaneous HRR in vivo in the absence of PARP1 using the pun assay. Furthermore, we found that the HRR events that occur in Parp1 nullizygous mice are associated with a significant increase in large, clonal events, as opposed to the usually more frequent single cell events, suggesting an effect in replicating cells. In conclusion, our data demonstrates that PARP1 inhibits spontaneous HRR events, and supports the model of DNA replication transformation of SSBs into HRR substrates.

Changes of senescent cell accumulation and removal in skin tissue with ageing.

Cellular senescence prevents the proliferation of cells at risk for neoplastic transformation. Nonetheless, the senescence response is thought to be antagonistically pleiotropic and thus contribute to aging phenotypes, including, ironically, late life cancers. The cancer-promoting activity of senescent cells is likely due to secreted molecules, the identity of which remains largely unknown. Here, we have shown that senescent fibroblasts, much more than presenescent fibroblasts, stimulate tumor vascularization in mice. Weakly malignant epithelial cells co-injected with senescent fibroblasts had larger and greater numbers of blood vessels compared with controls. Accordingly, increased vascular endothelial growth factor (VEGF) expression was a frequent characteristic of senescent human and mouse fibroblasts in culture. Importantly, conditioned medium from senescent fibroblasts, more than medium from presenescent cells, stimulates cultured human umbilical vein endothelial cells to invade a basement membrane, a hallmark of angiogenesis. Increased VEGF expression was specific to the senescent phenotype and increased whether senescence was induced by replicative exhaustion, overexpression of p16(Ink4a), or overexpression of oncogenic RAS. The senescence-dependent increase in VEGF production was accompanied by very little increase in hypoxic-inducible (transcription) factor 1 alpha protein levels, and hypoxia further induced VEGF in senescent cells. This result suggests the rise in VEGF expression at senescence is not a hypoxic response. Our findings may in part explain why senescent cells stimulate tumorigenesis in vivo and support the idea that senescent cells may facilitate age-associated cancer development by secreting factors that promote malignant progression.

Cancer progression is supported by the cross-talk between tumor cells and the surrounding stroma. In this context, senescent cells in the tumor microenvironment contribute to the development of a pro-inflammatory milieu and the acquisition of aggressive traits by cancer cells. Anticancer treatments induce cellular senescence (therapy-induced senescence, TIS) in both tumor and non-cancerous cells, contributing to many detrimental side effects of therapies. Thus, we focused on the effects of chemotherapy on the stromal compartment of prostate and ovarian cancer. We demonstrated that anticancer chemotherapeutics, regardless of their specific mechanism of action, promote a senescent phenotype in stromal fibroblasts, resulting in metabolic alterations and secretion of paracrine factors, sustaining the invasive and clonogenic potential of both prostate and ovarian cancer cells. The clearance of senescent stromal cells, through senolytic drug treatment, reverts the malignant phenotype of tumor cells. The clinical relevance of TIS was validated in ovarian and prostate cancer patients, highlighting increased accumulation of lipofuscin aggregates, a marker of the senescent phenotype, in the stromal compartment of tissues from chemotherapy-treated patients. These data provide new insights into the potential efficacy of combining traditional anticancer strategies with innovative senotherapy to potentiate anticancer treatments and overcome the adverse effects of chemotherapy.

Senescent cell accumulation is a known driver of aging and age-related pathologies. Clearance of senescent cells is a promising approach to increase longevity and reduce multiple age-related diseases in humans. Flavonoid compounds are present in fruits and vegetables, of which certain flavonoids (e.g., quercetin and fisetin) have demonstrated senolytic, or ‘senescent-cell-killing’, activity in culture and in mice. However, the cellular mechanism of senolysis and the efficacy of these compounds are not known in renal epithelial cells. Here, we combine flavonoid drug screening, targeted metabolomics, and thermal proteome profiling (TPP) with analysis by mass spectrometry (MS) to explore protein targets of senolytic drugs in senescent human renal cortical epithelial cells and human renal proximal tubular epithelial cells. Senescence was induced by exposure to ionizing radiation and the senescence phenotype was validated through a rigorous panel of senescence and viability markers. Proliferating and senescent renal epithelial cells were screened with a panel of 8 flavonoids to identify senolytic drugs and their concentrations for selectively killing senescent cells. Targeted MS assays were developed to assess intracellular drug uptake, a potential mechanism of senescent-specific killing. To identify protein targets of the senolytic flavonoids in senescent cells, we performed a variation of TPP in combination with liquid chromatography (LC)-MS/MS proteomics analysis, carefully controlling for non-senolytic interactions by excluding proteins bound by a non-senolytic flavonoid. The results of this study pave the way for the development of a more specific generation of senolytic compounds and identify novel candidate senolytic pathways engaged by dietary flavonoids.

Poly (ADP‒ribose) polymerase inhibitors (PARPis) are widely used in maintenance therapy for various platinum-sensitive cancers regardless of the occurrence of BRCA mutations. However, the mechanisms of action and treatment resistance associated with the use of PARPis for maintenance therapy in pancreatic cancer remain unclear. In this study, in addition to the induction of apoptosis, the use of PARPis (olaparib and niraparib) as maintenance therapies inhibited cell proliferation by causing cellular senescence to exert potent anticancer effects on Capan-1 (BRCA mutated) and PANC-1 (BRCA wild-type) cells. Mechanistically, the cellular senescence caused by PARPis relies on the Chk2‒p21 pathway but not in a p53-dependent manner. Interestingly, in addition to directly causing DNA damage, PARPis also exacerbate DNA damage through the generation of ROS via the positive feedback pathway, thereby inducing cellular senescence. Unfortunately, PARPis therapy-induced senescence is a reversible anticancer mechanism in which senescent cancer cells lose their senescence-like phenotype and continue proliferating upon drug withdrawal. This potentially explains the requirement for sustained PARPi therapy in the clinic. Furthermore, the expression of Bcl-2 was increased in PARPi-induced senescent cancer cells, providing a window for opportunistic elimination via synergistic senolytic drugs. The inhibition of Bcl-2 through the sequence-dependent combination of navitoclax enhanced the anticancer effects of PARPis by removing senescent cells. Collectively, data from our study demonstrate that the clinical application of PARPis as maintenance therapy could be achieved through the induction of cellular senescence. Furthermore, sequence-dependent combination with senescence-targeting drugs can potentiate pancreatic cancer treatment effects of PARPis regardless of the BRCA status.

Intratumoral electroporation of IL12-encoding plasmids (IT-pIL12-EP) enhances antitumor immunity in treated and distant tumors. However, IT-pIL12-EP outcomes are in part dictated by infiltrating T-cell composition, where higher proportions of cytotoxic T cells as opposed to more immunosuppressive immune cells predicts enhanced IT-pIL12-EP responsiveness. In an effort to increase proportions of activated cytotoxic T cells in IT-pIL12-EP-treated tumors, Han and colleagues supplemented IT-pIL12-EP with electroporation of plasmids encoding membrane-anchored anti-CD3 single-chain variable fragments (scFv). The authors found that including anti-CD3 scFv with IL12 enhances proliferation and interferon-γ (IFNγ) secretion in both CD8+ cytotoxic T cells and CD4+ T regulatory (Treg) cells, and that costimulation with membrane-bound anti-CD3 with IL12 reduces Treg suppression. Supplementing IT-pIL12-EP with plasmids encoding anti-CD3 scFv in vivo enhances proliferation and cytotoxicity of tumor antigen-specific T cells, leading to augmented antitumor immunity against B16-F10 melanoma and 4T1 mammary tumor cells. Treating human melanoma-infiltrating T cells with IL12 and anti-CD3 scFv enhances cytotoxic CD8+ expansion, IFNγ secretion, and PD-1 expression in vitro, suggesting IT-pIL12-EP and anti-CD3 scFv could augment PD-1 immune checkpoint inhibition responsiveness in melanoma patients.Tumor necrosis factor alpha (TNFα)-mediated cytotoxicity can be resisted via NF-κB activation and downstream survival signals in head and neck squamous cell carcinoma (HNSCC). Toxicities accompanying NF-κB signaling inhibition necessitate other methods of targeting the pathway. To search for novel targets affecting NF-κB signaling, Hu and colleagues performed a RNAi screen targeting the kinome and druggable genomic targets in a HNSCC cell line expressing NF-κB promoter response elements in tandem with a β-lactamase reporter gene. The screen revealed that G2/M checkpoint kinases WEE1 and cyclin dependent kinase 1 (CDK1) promote NF-κB signaling. WEE1 inhibition using AZD1775 disrupts WEE1 interactions with IκB kinase (IKK)α/β and RELA, reducing IKKα/β and RELA phosphorylation and NF-κB signaling. Correspondingly, AZD1775 augments TNFα-mediated HNSCC cell death, including in HNSCC radiotherapy in vivo. WEE1 and CDK1 expression levels and disease outcome correlations are dependent on human papillomavirus (HPV) infection status, suggesting HPV influences the signaling pathway. In addition to unveiling a novel signaling axis, this study presents a new potential way in which NF-κB can be therapeutically targeted in HNSCC.Decreased SIN3A expression in human breast cancer cell lines enhances tumorigenic phenotypes in vitro and tumor progression in vivo. However, how SIN3A abundance in breast tumors compares to its abundance in normal breast tissue is unknown, as is how SIN3A expression is regulated in breast tumors. To address these questions, Davenport and colleagues assessed SIN3A mRNA and corresponding protein levels in patients and validated these findings using public data sets (TCGA and METABRIC). The authors found that the SIN3A mRNA 3′ untranslated region harbors a sequence complementary to that of oncogenic miR-183, and that miR-183 silences SIN3A expression and enhances expression of genes that SIN3A transcriptionally represses. Accordingly, miR-183 expression negatively correlates with breast cancer patient survival and induces breast cancer cell migration and invasion in vitro and pulmonary metastasis in vivo through SIN3A downregulation. Overall, the novel miR-183-mediated regulation of SIN3A expression uncovered in this study could be used to inform metastatic breast cancer intervention strategies moving forward.Radiation and temozolomide (TMZ), the standard treatments for glioblastoma (GBM), are known to induce a non-proliferative, senescent phenotype in surviving GBM cells. However, GBM cells often eventually escape treatment-induced senescence, leading to residual tumor outgrowth and GBM recurrence. To assess whether recently discovered senolytic drugs can eliminate senescent GBM cells, Rahman and colleagues tested the effects of 10 senolytic drugs on 12 human GBM cell lines treated with radiation or TMZ. The authors found that senolytic drugs targeting BCL-XL, as well as siRNA-mediated BCL-XL abrogation, enhance senescent GBM cell death. The results of the study suggest that pharmacological BCL-XL inhibition could augment antitumor effects of radiation and TMZ treatment, and potentially inhibit residual GBM cell outgrowth and disease recurrence.

Aging and cancer share overlapping characteristics, referred to as meta-hallmarks, which elucidate the convergent, antagonistic, or contradictory relationships between aging and cancer. Likewise, as a key characteristic of aging, senescent cells share some meta-hallmarks with tumor cells. These hallmarks include apoptosis resistance, metabolic alterations, secretory phenotypes, epigenetic reprogramming, and immune surveillance, all of which play pivotal roles in both tumorigenesis and senescence. Moreover, senolytic drugs, which are a class of agents selectively designed to eliminate senescent cells, have emerged as promising therapeutic agents in oncology and aging-related diseases. Since the discovery of the first senolytic drug in 2015, a diverse array of such agents has been developed. Notably, most senolytic drugs are repurposed from existing anti-tumor therapies, leveraging their shared mechanisms with senescent cells and tumor cells. Thus, this review examines the similarities between senescent cells and tumor cells, providing a better understanding of the meta-hallmarks. Besides, we categorize existing senolytic drugs based upon meta-hallmarks and elucidate the potential molecular mechanisms underlying their effects. By integrating insights from cancer and senescence research, this work aims to inspire innovative strategies for senolytic drug discovery.

Malignant Melanoma that resists immunotherapy remains the deadliest form of skin cancer owing to poor clinically lasting responses. Alternative like genotoxic or targeted chemotherapy trigger various cancer cell fates after treatment including cell death and senescence. Senescent cells can be eliminated using senolytic drugs and we hypothesize that the targeted elimination of therapy-induced senescent melanoma cells could complement both conventional and immunotherapies. We utilized a panel of cells representing diverse mutational background relevant to melanoma and found that they developed distinct senescent phenotypes in response to treatment. A genotoxic combination therapy of carboplatin-paclitaxel or irradiation triggered a mixed response of cell death and senescence, irrespective of BRAF mutation profiles. DNA damage-induced senescent melanoma cells exhibited morphological changes, residual DNA damage, and increased senescence-associated secretory phenotype (SASP). In contrast, dual targeted inhibition of Braf and Mek triggered a different mixed cell fate response including senescent-like and persister cells. While persister cells could reproliferate, senescent-like cells were stably arrested, but without detectable DNA damage and senescence-associated secretory phenotype. To assess the sensitivity to senolytics we employed a novel real-time imaging-based death assay and observed that Bcl2/Bcl-XL inhibitors and piperlongumine were effective in promoting death of carboplatin-paclitaxel and irradiation-induced senescent melanoma cells, while the mixed persister cells and senescent-like cells resulting from Braf-Mek inhibition remained unresponsive. Interestingly, a direct synergy between Bcl2/Bcl-XL inhibitors and Braf-Mek inhibitors was observed when used out of the context of senescence. Overall, we highlight diverse hallmarks of melanoma senescent states and provide evidence of context-dependent senotherapeutics that could reduce treatment resistance while also discussing the limitations of this strategy in human melanoma cells.