RESUMO
PIM kinases are implicated in variety of cancers by promoting cell survival and proliferation and are targets of interest for therapeutic intervention. We have identified a low-nanomolar pan-PIM inhibitor (PIM1/2/3 potency 5:14:2nM) using structure based modeling. The crystal structure of this compound with PIM1 confirmed the predicted binding mode and protein-ligand interactions except those in the acidic ribose pocket. We show the SAR suggesting the importance of having a hydrogen bond donor in this pocket for inhibiting PIM2; however, this interaction is not important for inhibiting PIM1 or PIM3. In addition, we report the discovery of a new class of PIM inhibitors by using computational de novo design tool implemented in MOE software (Chemical Computing Group). These inhibitors have a different interaction profile.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Ligação Proteica , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
The DNA damage response (DDR) pathway regulates DNA repair and cell survival, and inactivating mutations in DDR genes can increase tumour mutational burden (TMB), a predictive biomarker of treatment benefit from anti-PD-1/PD-L1 immunotherapies. However, a better understanding of the relationship among specific DDR mutations, TMB and PD-L1 expression is needed to improve translational strategies. Here, we determined genomic alteration frequencies in selected DDR genes that are clinically actionable biomarkers and investigated their association with TMB and PD-L1 in bladder, colorectal, non-small cell lung, ovarian and prostate cancers using the FoundationInsights® web portal. Our results not only confirm known associations, such as mismatch repair and POLE gene mutations with high TMB, but also identify significant associations between mutations in the SWI/SNF chromatin remodelling genes ARID1A and SMARCA4 and high TMB in multiple tumour types. Mutations in the ATR gene were associated with high TMB in colorectal and prostate cancers; however, associations between individual DDR mutations and high PD-L1 expression were uncommon and tumour-type specific. Finally, we found that high TMB and high PD-L1 expression were poorly associated, emphasising their independence as predictive biomarkers for immune checkpoint inhibitor use.
RESUMO
Ataxia telangiectasia and Rad3-related protein (ATR) kinase regulate a key cell regulatory node for maintaining genomic integrity by preventing replication fork collapse. ATR inhibition has been shown to increase replication stress resulting in DNA double-strand breaks (DSBs) and cancer cell death, and several inhibitors are under clinical investigation for cancer therapy. However, activation of cell-cycle checkpoints controlled by ataxia telangiectasia-mutated (ATM) kinase could minimize the lethal consequences of ATR inhibition and protect cancer cells. Here, we investigate ATR-ATM functional relationship and potential therapeutic implications. In cancer cells with functional ATM and p53 signaling, selective suppression of ATR catalytic activity by M6620 induced G1-phase arrest to prevent S-phase entry with unrepaired DSBs. The selective ATM inhibitors, M3541 and M4076, suppressed both ATM-dependent cell-cycle checkpoints, and DSB repair lowered the p53 protective barrier and extended the life of ATR inhibitor-induced DSBs. Combination treatment amplified the fraction of cells with structural chromosomal defects and enhanced cancer cell death. ATM inhibitor synergistically potentiated the ATR inhibitor efficacy in cancer cells in vitro and increased ATR inhibitor efficacy in vivo at doses that did not show overt toxicities. Furthermore, a combination study in 26 patient-derived xenograft models of triple-negative breast cancer with the newer generation ATR inhibitor M4344 and ATM inhibitor M4076 demonstrated substantial improvement in efficacy and survival compared with single-agent M4344, suggesting a novel and potentially broad combination approach to cancer therapy.
Assuntos
Ataxia Telangiectasia , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteínas Mutadas de Ataxia Telangiectasia , Reparo do DNA , Proteínas de Ciclo Celular/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Dano ao DNA , Quinase 1 do Ponto de Checagem/genéticaRESUMO
The PI3K-Akt-mTOR (PAM) pathway is implicated in tumor progression in many tumor types, including metastatic gastric cancer (GC). The initial promise of PAM inhibitors has been unrealized in the clinic, presumably due, in part, to the up-regulation of Akt signaling that occurs when the pathway is inhibited. Here we present that DIACC3010 (formerly M2698), an inhibitor of two nodes in the PAM pathway, p70S6K and Akt 1/3, blocks the pathway in in vitro and in vivo preclinical models of GC while providing a mechanism that inhibits signaling from subsequent Akt up-regulation. Utilizing GC cell lines and xenograft models, we identified potential markers of DIACC3010-sensitivity in Her2-negative tumors, i.e., PIK3CA mutations, low basal pERK, and a group of differentially expressed genes (DEGs). The combination of DIACC3010 and trastuzumab was evaluated in Her2-positive cell lines and models. Potential biomarkers for the synergistic efficacy of the combination of DIACC3010 + trastuzumab also included DEGs as well as a lack of up-regulation of pERK. Of 27 GC patient-derived xenograft (PDX) models tested in BALB/c nu/nu mice, 59% were sensitive to DIACC3010 + trastuzumab. Of the 21 HER2-negative PDX models, DIACC3010 significantly inhibited the growth of 38%. Altogether, these results provide a path forward to validate the potential biomarkers of DIACC3010 sensitivity in GC and support clinical evaluation of DIACC3010 monotherapy and combination with trastuzumab in patients with HER2- negative and positive advanced GCs, respectively.
Assuntos
Neoplasias Gástricas , Animais , Camundongos , Humanos , Neoplasias Gástricas/tratamento farmacológico , Proteínas Quinases S6 Ribossômicas 70-kDa , Proteínas Proto-Oncogênicas c-akt , Fosfatidilinositol 3-Quinases , Inibidores de Proteínas Quinases , Inibidores da Angiogênese , Modelos Animais de DoençasRESUMO
Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Specifically, MYC-driven non-neuroendocrine SCLC is particularly resistant to standard therapies. Lurbinectedin was recently approved for the treatment of relapsed SCLC, but combinatorial approaches are needed to increase the depth and duration of responses to lurbinectedin. Using high-throughput screens, we found inhibitors of ataxia telangiectasia mutated and rad3 related (ATR) as the most effective agents for augmenting lurbinectedin efficacy. First-in-class ATR inhibitor berzosertib synergized with lurbinectedin in multiple SCLC cell lines, organoid, and in vivo models. Mechanistically, ATR inhibition abrogated S-phase arrest induced by lurbinectedin and forced cell cycle progression causing mitotic catastrophe and cell death. High CDKN1A/p21 expression was associated with decreased synergy due to G1 arrest, while increased levels of ERCC5/XPG were predictive of increased combination efficacy. Importantly, MYC-driven non-neuroendocrine tumors which are resistant to first-line therapies show reduced CDKN1A/p21 expression and increased ERCC5/XPG indicating they are primed for response to lurbinectedin-berzosertib combination. The combination is being assessed in a clinical trial NCT04802174.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma de Pequenas Células do Pulmão , Humanos , Recidiva Local de Neoplasia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Neoplasias Pulmonares/patologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
Small cell neuroendocrine cancers (SCNCs) are recalcitrant cancers arising from diverse primary sites that lack effective treatments. Using chemical genetic screens, we identified inhibition of ataxia telangiectasia and rad3 related (ATR), the primary activator of the replication stress response, and topoisomerase I (TOP1), nuclear enzyme that suppresses genomic instability, as synergistically cytotoxic in small cell lung cancer (SCLC). In a proof-of-concept study, we combined M6620 (berzosertib), first-in-class ATR inhibitor, and TOP1 inhibitor topotecan in patients with relapsed SCNCs. Objective response rate among patients with SCLC was 36% (9/25), achieving the primary efficacy endpoint. Durable tumor regressions were observed in patients with platinum-resistant SCNCs, typically fatal within weeks of recurrence. SCNCs with high neuroendocrine differentiation, characterized by enhanced replication stress, were more likely to respond. These findings highlight replication stress as a potentially transformative vulnerability of SCNCs, paving the way for rational patient selection in these cancers, now treated as a single disease.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Isoxazóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Recidiva Local de Neoplasia/tratamento farmacológico , Pirazinas/farmacologia , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Idoso , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Replicação do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo I/genética , Instabilidade Genômica/genética , Humanos , Neoplasias Pulmonares/metabolismo , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Carcinoma de Pequenas Células do Pulmão/metabolismoRESUMO
Targeting the DNA damage response (DDR) in tumors with defective DNA repair is a clinically successful strategy. The RAS/RAF/MEK/ERK signalling pathway is frequently deregulated in human cancers. In this study, we explored the effects of MEK inhibition on the homologous recombination pathway and explored the potential for combination therapy of MEK inhibitors with DDR inhibitors and a hypoxia-activated prodrug. We studied effects of combining pimasertib, a selective allosteric inhibitor of MEK1/2, with olaparib, a small molecule inhibitor of poly (adenosine diphosphate [ADP]-ribose) polymerases (PARP), and with the hypoxia-activated prodrug evofosfamide in ovarian and pancreatic cancer cell lines. Apoptosis was assessed by Caspase 3/7 assay and protein expression was detected by immunoblotting. DNA damage response was monitored with γH2AX and RAD51 immunofluorescence staining. In vivo antitumor activity of pimasertib with evofosfamide were assessed in pancreatic cancer xenografts. We found that BRCA2 protein expression was downregulated following pimasertib treatment under hypoxic conditions. This translated into reduced homologous recombination repair demonstrated by levels of RAD51 foci. MEK inhibition was sufficient to induce formation of γH2AX foci, suggesting that inhibition of this pathway would impair DNA repair. When combined with olaparib or evofosfamide, pimasertib treatment enhanced DNA damage and increased apoptosis. The combination of pimasertib with evofosfamide demonstrated increased anti-tumor activity in BRCA wild-type Mia-PaCa-2 xenograft model, but not in the BRCA mutated BxPC3 model. Our data suggest that targeted MEK inhibition leads to impaired homologous recombination DNA damage repair and increased PARP inhibition sensitivity in BRCA-2 proficient cancers.
RESUMO
While it is clear that cancer arises from the accumulation of genetic mutations that endow the malignant cell with the properties of uncontrolled growth and proliferation, the precise combinations of mutations that program human tumor cell growth remain unknown. The study of the transforming proteins derived from DNA tumor viruses in experimental models of transformation has provided fundamental insights into the process of cell transformation. We recently reported that coexpression of the simian virus 40 (SV40) early region (ER), the gene encoding the telomerase catalytic subunit (hTERT), and an oncogenic allele of the H-ras gene in normal human fibroblast, kidney epithelial, and mammary epithelial cells converted these cells to a tumorigenic state. Here we show that the SV40 ER contributes to tumorigenic transformation in the presence of hTERT and oncogenic H-ras by perturbing three intracellular pathways through the actions of the SV40 large T antigen (LT) and the SV40 small t antigen (ST). LT simultaneously disables the retinoblastoma (pRB) and p53 tumor suppressor pathways; however, complete transformation of human cells requires the additional perturbation of protein phosphatase 2A by ST. Expression of ST in this setting stimulates cell proliferation, permits anchorage-independent growth, and confers increased resistance to nutrient deprivation. Taken together, these observations define the elements of the SV40 ER required for the transformation of human cells and begin to delineate a set of intracellular pathways whose disruption, in aggregate, appears to be necessary to generate tumorigenic human cells.
Assuntos
Antígenos Virais de Tumores/metabolismo , Transformação Celular Neoplásica , Vírus 40 dos Símios/fisiologia , Antígenos Virais de Tumores/genética , Divisão Celular , Linhagem Celular , Senescência Celular , Proteínas de Ligação a DNA , Fibroblastos , Humanos , Proteína Oncogênica p21(ras)/genética , Proteína Oncogênica p21(ras)/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Vírus 40 dos Símios/genética , Telomerase/genética , Telomerase/metabolismo , Fatores de Tempo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
Genetic changes in epithelial cells initiate the development of prostatic adenocarcinomas. As nascent tumors grow and undergo progression, epithelial tumor cells are intimately associated with stromal cells. Stromal cells within the tumor microenvironment acquire new properties, including the capacity to promote phenotypic and genetic progression in adjacent epithelial cells. Affymetrix microarrays were used to identify 119 genes differentially expressed between normal-derived and carcinoma-derived prostatic stromal cells. These included 31 genes encoding extracellular proteins that may act as stromal-to-epithelial paracrine signals. Further investigation of one of these genes, secreted frizzled related protein 1 (SFRP1), revealed that its expression parallels prostatic growth with high expression during prostatic development, low expression in the adult prostate, and elevated expression in prostatic tumor stroma. In addition, as prostatic epithelial cells progressed to a tumorigenic state under the influence of tumor stroma, SFRP1 became overexpressed in the progressed epithelial cells. To further understand the roles of SFRP1 in the prostate, we tested the affects of increased SFRP1 levels on prostatic tissues and cells. Treatment of developing prostates with SFRP1 in culture led to increased organ growth. Treatment of a human prostatic epithelial cell line with SFRP1 led to increased proliferation, decreased apoptosis, and decreased signaling through the Wnt/beta-catenin pathway in vitro and increased proliferation in vivo. These data suggest that overexpression of SFRP1 by prostatic tumor stroma may account for the previously reported capacity of prostatic tumor stroma to provide a pro-proliferative paracrine signal to adjacent epithelial cells.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Animais , Apoptose/genética , Processos de Crescimento Celular/genética , Progressão da Doença , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Neoplasias da Próstata/metabolismo , Transdução de Sinais/genética , Células Estromais/metabolismo , Células Estromais/patologia , Transfecção , Células Tumorais Cultivadas , Proteínas Wnt/biossíntese , Proteínas Wnt/genética , beta Catenina/biossíntese , beta Catenina/genéticaRESUMO
One critical step in the development of a cancerous cell is its acquisition of an unlimited replicative lifespan, the process termed immortalization. Experimental model systems designed to study cellular transformation ex vivo have relied to date on the in vitro selection of a subpopulation of cells that have become immortalized through treatment with chemical or physical mutagens and the selection of rare clonal variants. In this study, we describe the direct immortalization of primary human airway epithelial cells through the successive introduction of the Simian Virus 40 Early Region and the telomerase catalytic subunit hTERT. Cells immortalized in this way are now responsive to malignant transformation by an introduced H-ras or K-ras oncogene. These immortalized human airway epithelial cells, which have been created through the stepwise introduction of genetic alterations, provide a novel experimental model system with which to study further the biology of the airway epithelial cell and to dissect the molecular basis of lung cancer pathogenesis.