RESUMO
PURPOSE: We evaluated the properties and activity of AZD9574, a blood-brain barrier (BBB) penetrant selective inhibitor of PARP1, and assessed its efficacy and safety alone and in combination with temozolomide (TMZ) in preclinical models. EXPERIMENTAL DESIGN: AZD9574 was interrogated in vitro for selectivity, PARylation inhibition, PARP-DNA trapping, the ability to cross the BBB, and the potential to inhibit cancer cell proliferation. In vivo efficacy was determined using subcutaneous as well as intracranial mouse xenograft models. Mouse, rat, and monkey were used to assess AZD9574 BBB penetration and rat models were used to evaluate potential hematotoxicity for AZD9574 monotherapy and the TMZ combination. RESULTS: AZD9574 demonstrated PARP1-selectivity in fluorescence anisotropy, PARylation, and PARP-DNA trapping assays and in vivo experiments demonstrated BBB penetration. AZD9574 showed potent single agent efficacy in preclinical models with homologous recombination repair deficiency in vitro and in vivo. In an O6-methylguanine-DNA methyltransferase (MGMT)-methylated orthotopic glioma model, AZD9574 in combination with TMZ was superior in extending the survival of tumor-bearing mice compared with TMZ alone. CONCLUSIONS: The combination of three key features-PARP1 selectivity, PARP1 trapping profile, and high central nervous system penetration in a single molecule-supports the development of AZD9574 as the best-in-class PARP inhibitor for the treatment of primary and secondary brain tumors. As documented by in vitro and in vivo studies, AZD9574 shows robust anticancer efficacy as a single agent as well as in combination with TMZ. AZD9574 is currently in a phase I trial (NCT05417594). See related commentary by Lynce and Lin, p. 1217.
Assuntos
Neoplasias Encefálicas , Glioma , Animais , Humanos , Camundongos , Ratos , Antineoplásicos Alquilantes/farmacologia , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , DNA , Glioma/tratamento farmacológico , Glioma/patologia , O(6)-Metilguanina-DNA Metiltransferase/genética , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: We hypothesized that inhibition and trapping of PARP1 alone would be sufficient to achieve antitumor activity. In particular, we aimed to achieve selectivity over PARP2, which has been shown to play a role in the survival of hematopoietic/stem progenitor cells in animal models. We developed AZD5305 with the aim of achieving improved clinical efficacy and wider therapeutic window. This next-generation PARP inhibitor (PARPi) could provide a paradigm shift in clinical outcomes achieved by first-generation PARPi, particularly in combination. EXPERIMENTAL DESIGN: AZD5305 was tested in vitro for PARylation inhibition, PARP-DNA trapping, and antiproliferative abilities. In vivo efficacy was determined in mouse xenograft and PDX models. The potential for hematologic toxicity was evaluated in rat models, as monotherapy and combination. RESULTS: AZD5305 is a highly potent and selective inhibitor of PARP1 with 500-fold selectivity for PARP1 over PARP2. AZD5305 inhibits growth in cells with deficiencies in DNA repair, with minimal/no effects in other cells. Unlike first-generation PARPi, AZD5305 has minimal effects on hematologic parameters in a rat pre-clinical model at predicted clinically efficacious exposures. Animal models treated with AZD5305 at doses ≥0.1 mg/kg once daily achieved greater depth of tumor regression compared to olaparib 100 mg/kg once daily, and longer duration of response. CONCLUSIONS: AZD5305 potently and selectively inhibits PARP1 resulting in excellent antiproliferative activity and unprecedented selectivity for DNA repair deficient versus proficient cells. These data confirm the hypothesis that targeting only PARP1 can retain the therapeutic benefit of nonselective PARPi, while reducing potential for hematotoxicity. AZD5305 is currently in phase I trials (NCT04644068).
Assuntos
Antineoplásicos , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Camundongos , Ratos , Animais , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Ftalazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1 , Antineoplásicos/farmacologia , Reparo do DNARESUMO
PARP inhibitors (PARPi) are currently indicated for the treatment of ovarian, breast, pancreatic, and prostate cancers harboring mutations in the tumor suppressor genes BRCA1 or BRCA2. In the case of ovarian and prostate cancers, their classification as homologous recombination repair (HRR) deficient (HRD) or mutated also makes PARPi an available treatment option beyond BRCA1 or BRCA2 mutational status. However, identification of the most relevant genetic alterations driving the HRD phenotype has proven difficult and recent data have shown that other genetic alterations not affecting HRR are also capable of driving PARPi responses. To gain insight into the genetics driving PARPi sensitivity, we performed CRISPR-Cas9 loss-of-function screens in six PARPi-insensitive cell lines and combined the output with published PARPi datasets from eight additional cell lines. Ensuing exploration of the data identified 110 genes whose inactivation is strongly linked to sensitivity to PARPi. Parallel cell line generation of isogenic gene knockouts in ovarian and prostate cancer cell lines identified that inactivation of core HRR factors is required for driving in vitro PARPi responses comparable with the ones observed for BRCA1 or BRCA2 mutations. Moreover, pan-cancer genetic, transcriptomic, and epigenetic data analyses of these 110 genes highlight the ones most frequently inactivated in tumors, making this study a valuable resource for prospective identification of potential PARPi-responsive patient populations. Importantly, our investigations uncover XRCC3 gene silencing as a potential new prognostic biomarker of PARPi sensitivity in prostate cancer. Significance: This study identifies tumor genetic backgrounds where to expand the use of PARPis beyond mutations in BRCA1 or BRCA2. This is achieved by combining the output of unbiased genome-wide loss-of-function CRISPR-Cas9 genetic screens with bioinformatics analysis of biallelic losses of the identified genes in public tumor datasets, unveiling loss of the DNA repair gene XRCC3 as a potential biomarker of PARPi sensitivity in prostate cancer.
Assuntos
Neoplasias Ovarianas , Neoplasias da Próstata , Humanos , Masculino , Feminino , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Estudos Prospectivos , Neoplasias da Próstata/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , BiomarcadoresRESUMO
The receptor-interacting serine/threonine protein kinase 1 (RIPK1) is a key mediator of regulated cell death and inflammation. Recent studies suggest that RIPK1 inhibition would fundamentally improve the therapy of RIPK1-dependent organ damage in stroke, myocardial infarction, kidney failure, and systemic inflammatory response syndrome. Additionally, it could ameliorate or prevent multi-organ failure induced by cytokine release in the context of hyperinflammation, as seen in COVID-19 patients. Therefore, we searched for a RIPK1 inhibitor and present the aromatic antiepileptic and FDA-approved drug primidone (Liskantin®) as a potent inhibitor of RIPK1 activation in vitro and in a murine model of TNFα-induced shock, which mimics the hyperinflammatory state of cytokine release syndrome. Furthermore, we detected for the first time RIPK1 activation in the respiratory tract epithelium of hospitalized patients who tested positive for SARS-CoV-2 infection. Our data provide a strong rationale for evaluating the drug primidone in conditions of hyperinflammation in humans.
Assuntos
COVID-19/enzimologia , Primidona/farmacologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , SARS-CoV-2/metabolismo , Animais , COVID-19/patologia , Morte Celular/efeitos dos fármacos , Células HEK293 , Células HT29 , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Inflamação/patologia , Células Jurkat , Camundongos , Células NIH 3T3 , Células U937 , Tratamento Farmacológico da COVID-19RESUMO
Drugs that mobilise the immune system against cancer are dramatically improving care for many people. Dying cancer cells play an active role in inducing anti-tumour immunity but not every form of death can elicit an immune response. Moreover, resistance to apoptosis is a major problem in cancer treatment and disease control. While the term "immunogenic cell death" is not fully defined, activation of receptor-interacting serine/threonine-protein kinase 1 (RIPK1) can induce a type of death that mobilises the immune system against cancer. However, no clinical treatment protocols have yet been established that would harness the immunogenic potential of RIPK1. Here, we report the first pre-clinical application of an in vivo treatment protocol for soft-tissue sarcoma that directly engages RIPK1-mediated immunogenic cell death. We find that RIPK1-mediated cell death significantly improves local disease control, increases activation of CD8+ T cells as well as NK cells, and enhances the survival benefit of immune checkpoint blockade. Our findings warrant a clinical trial to assess the survival benefit of RIPK1-induced cell death in patients with advanced disease at limb extremities.
Assuntos
Morte Celular Imunogênica , Sarcoma , Apoptose , Linfócitos T CD8-Positivos/metabolismo , Humanos , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Sarcoma/terapia , Transdução de Sinais , Fator de Necrose Tumoral alfaRESUMO
Tumor necrosis factor (TNF) is an inflammatory cytokine that can signal cell survival or cell death. The mechanisms that switch between these distinct outcomes remain poorly defined. Here, we show that the E3 ubiquitin ligase Mind Bomb-2 (MIB2) regulates TNF-induced cell death by inactivating RIPK1 via inhibitory ubiquitylation. Although depletion of MIB2 has little effect on NF-κB activation, it sensitizes cells to RIPK1- and caspase-8-dependent cell death. We find that MIB2 represses the cytotoxic potential of RIPK1 by ubiquitylating lysine residues in the C-terminal portion of RIPK1. Our data suggest that ubiquitin conjugation of RIPK1 interferes with RIPK1 oligomerization and RIPK1-FADD association. Disruption of MIB2-mediated ubiquitylation, either by mutation of MIB2's E3 activity or RIPK1's ubiquitin-acceptor lysines, sensitizes cells to RIPK1-mediated cell death. Together, our findings demonstrate that Mind Bomb E3 ubiquitin ligases can function as additional checkpoint of cytokine-induced cell death, selectively protecting cells from the cytotoxic effects of TNF.
Assuntos
Apoptose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Multimerização Proteica/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/efeitos dos fármacosRESUMO
TNF is an inflammatory cytokine that upon binding to its receptor, TNFR1, can drive cytokine production, cell survival, or cell death. TNFR1 stimulation causes activation of NF-κB, p38α, and its downstream effector kinase MK2, thereby promoting transcription, mRNA stabilization, and translation of target genes. Here we show that TNF-induced activation of MK2 results in global RIPK1 phosphorylation. MK2 directly phosphorylates RIPK1 at residue S321, which inhibits its ability to bind FADD/caspase-8 and induce RIPK1-kinase-dependent apoptosis and necroptosis. Consistently, a phospho-mimetic S321D RIPK1 mutation limits TNF-induced death. Mechanistically, we find that phosphorylation of S321 inhibits RIPK1 kinase activation. We further show that cytosolic RIPK1 contributes to complex-II-mediated cell death, independent of its recruitment to complex-I, suggesting that complex-II originates from both RIPK1 in complex-I and cytosolic RIPK1. Thus, MK2-mediated phosphorylation of RIPK1 serves as a checkpoint within the TNF signaling pathway that integrates cell survival and cytokine production.
Assuntos
Apoptose/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Caspase 8/metabolismo , Relação Dose-Resposta a Droga , Proteína de Domínio de Morte Associada a Fas/metabolismo , Células HT29 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos , NF-kappa B/metabolismo , Necrose , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/efeitos dos fármacos , TransfecçãoRESUMO
The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues.