Checkpoint Mitótico

Niara Oliveira

Definição

•É o principal mecanismo de controle do ciclo celular durante a mitose

•É o responsável pela produção de células-filhas geneticamente idênticas à progenitora pois assegura a adequada segregação cromossômica

•“Controle de qualidade” do ciclo celular

Checkpoint Mitótico

Mecanismo que bloqueia o início da anáfase e o subseqüente término da

mitose até que todos os cromossomos estejam adequadamente alinhados no

plano equatorial e ligados a microtúbulos de pólos opostos do fuso mitótico.

Mitose

•Cada cromossomo mitótico, composto por 2 pares de cromátides-irmãs, move-se para o centro da célula durante a prófase.

•Uma vez alinhados, a célula inicia a metáfase.

•Na anáfase, as cromátides se separam, dirigindo-se sincronicamente para cada um dos pólos opostos da célula.

•A separação em células-filhas chama-se citocinese.

Cinetócoro

•Durante a condensação da cromatina na prófase, um par de organelas chamado CINETÓCORO se forma em cada cromossomo na região centromérica

•O cinetócoro é o responsável pela ligação aos microtúbulos do fuso mitótico e pela sinalização para o checkpoint

Cinetócoro

Cinetócoro

•Cinetócoros não ligados aos microtúbulos ou aqueles com ligação instável sinalizam para a ativação do mecanismo de checkpoint

•Mesmo um único cinetócoro não ligado é capaz de inibir o início da anáfase

•Após a adequada ligação do cinetócoro ao microtúbulo correspondente, o sinal cessa e a anáfase progride normalmente

Coesão cromossômica•As duas cromátides de um cromossomo são

ligadas através de um complexo de proteínas denominado COESINAS

•No início da anáfase, esse complexo é destruído por uma protease chamada SEPARASE, permitindo a segregação cromossômica

•No entanto, até esse momento, as separases se mantém inativas através da ligação com proteínas denominadas SECURINAS

Anáfase

• Início da anáfase é regulado pela ativação de um complexo de proteínas chamado anaphase-promoting complex or cyclosome – APC/C

•Ativação do APC/C se dá através de sua ligação com Cdc20

•APC/C é o responsável pela ubiquitinação das securinas, liberando a separase

Regulação do APC/C

•Ligação do APC/C com proteínas do checkpoint inibe sua ativação pelo Cdc20

•APC/C inativo é incapaz de ativar as separases

•Cromátides-irmãs se mantém unidas

APC/C = Alvo através do qual checkpoint mitótico regula o ciclo

celular!

Proteínas do Checkpoint

•Mad (mitotic arrest deficient) – composta por MAD 1, 2 e 3

•Bub (budding uninhibited by benzimidazole) – composta por BUB 1 e 3

•Mps1

Proteínas do Checkpoint

•Mad2 e Mad3/BubR1 são inibidores diretos da atividade do APC/C

•Não se sabe se essas proteínas fazem parte de um único complexo inibitório ou atuam separadamente

Turning off the checkpoint

•Há dois modelos:- Tensão mecânica gerada pela ligação a

microtúbulos de pólos opostos modula a atividade das enzimas do checkpoint no cinetócoro

- Competição – ocupação dos locais de ligação pelos microtúbulos impede ligação das proteínas do checkpoint, cessando a sinalização

Final da Mitose

•Cdk1 é a principal kinase que mantém o estado mitótico

•É ativada no início da mitose através da ligação com ciclina B

Final da Mitose

•Ativação do APC/C inicia a anáfase e, ao mesmo tempo, induz a degradação da ciclina B

•A degradação da ciclina B inativa a Cdk1, levando ao término do processo mitótico

Implicações Clínicas

Instabilidade Cromossômica

•Erros no processo de segregação podem levar a perda ou ganho de cromossomos

ANEUPLOIDIA - resultando em morte celular ou crescimento celular desordenado

Falhas no mecanismo de CheckpointDurante a MITOSEMITOSE

- perdas momentâneas em mitoses pós-zigóticas podem levar mosaicismo cromossômico

- perdas definitivas em mitoses somáticas podem desencadear processo neoplásico

Falhas no mecanismo de CheckpointDurante a MEIOSEMEIOSE

- A inativação do checkpoint durante o processo meiótico implica na formação de gametas portadores de aneuploidias cromossômicas

Checkpoint Mitótico e Câncer

Checkpoint Deficiente x Inativo

•Células com mecanismo deficiente de checkpoint interrompem anáfase pela sinalização dos cinetócoros, mas não conseguem evitar segregação cromossômica incorreta

- Algumas células necessitam número muito grande de cinetótoros não-ligados para que haja recrutamento de níveis ótimos de proteínas do checkpoint

Checkpoint Deficiente x Inativo

•Células com mecanismo de checkpoint inativo (ou completamente danificado) apresentam segregação cromossômica aberrante

- Um número mínimo de cromossomos/genes é necessário para a viabilidade celular

Checkpoint Deficiente x Inativo

•Checkpoint deficiente INSTABILIDADE CROMOSSÔMICA

•Checkpoint inativo MORTE CELULAR

Célula Neoplásica

•Mecanismo de tumorigênese está relacionado à presença do mecanismo deficiente do checkpoint mitótico

•Fraca sinalização do checkpoint na célula neoplásica é suficiente para manter a viabilidade celular sem, no entanto, prevenir contra a segregação cromossômica incorreta

Aneuploidia e Instabilidade Cromossômica

Checkpoint e Apoptose•Estudos sugerem ligação estreita entre

mecanismo de checkpoint e indução de apoptose

•No entanto, as moléculas que estabelecem essa relação permanecem desconhecidas

•Pesquisas com células neoplásicas mostram que mecanismo de checkpoint ativo é necessário para que ocorra morte celular programada

Checkpoint e Quimioterapia

•Taxanos (paclitaxel e docetaxel) estabilizam os microtúbulos, induzindo fuso multipolar

•Alcalóides da vinca (vimblastina e vincristina) inibem formação dos microtúbulos

Ambos desencadeiam mecanismo de checkpoint, interrompendo a divisão celular em anáfase

Checkpoint e Quimioterapia

Checkpoint e Quimioterapia

•Tratamento contínuo com essas medicações mantém ciclo celular parado

•A relação do checkpoint com o processo de apoptose é responsável pelo desencadeamento da morte celular

Checkpoint e Resistência à QT

•Células neoplásicas com mecanismo de checkpoint inativo são resistentes ao tratamento com taxanos e alcalóides da vinca

Checkpoint e Câncer

[586] Prospective study of changes in spindle assembly checkpoint (SAC) to predict breast tumor response to taxanes.

N. T. Ueno, S. Kim, W. F. Symmans, M. A. Detry, L. Pusztai, L. F. Sanchez, H. Ishihara, G. N. Hortobagyi, S. Noguchi. M. D. Anderson Cancer Center, Houston, TX, United States

Background: Increased cyclin-dependent kinase 1 (CDK1) and reduced CDK2 activity reflects taxane chemosensitivity via regulation of SAC. Novel C2P device (Sysmex Inc.) measures CDK1/2 activity in human tissue within a few hours. We hypothesized that SAC functionality in breast cancer predicts taxane sensitivity. Methods: Biopsy samples obtained before preoperative therapy from 69 pts with

primary breast cancer (median age 52, St II, 56%, St III, 40%, HER2 36%, HR 52%) ex vivo with paclitaxel (100 nM) for 24 h, then measured for CDK1/2 activity. Tumors with CDK1/2 ratio > 0.7 were

denoted "responders" (CR, PR) based on preclinical testing. Clinical responses were evaluated with sonography. Results: 24 pts could not be evaluated because of unmeasurable or invalid CDK (n=3), no

receipt of taxane (n=10), incomplete treatment/lack of imaging (n=10), or receipt of taxane as adjuvant therapy (n=1). Among the 45 evaluable pts, 30 obtained reading to determine the C2P-predicted response, which was significantly associated with the clinical response to preoperative therapy with paclitaxel, docetaxel, docetaxel/doxorubicin, paclitaxel/trastuzumab or lapatinib, or

docetaxel/capecitabine (P=0.002; sensitivity 0.83; specificity 0.86; positive predictive value [PPV] 0.95; and negative predictive value [NPV] 0.60). For HER2- tumors, the sensitivity was 0.94; specificity 0.83;

PPV 0.94; and NPV 0.83. We fit a series of logistic regression models to assess the relationship between C2P predicted response and clinical outcome. Each model included 1 clinical prognostic

factor (HR status, stage, NG, histology, HER2) as well as the C2P prediction factor. In all 5 models C2P remained a statistically significant predictor of clinical response while adjusting for the other

prognostic factor. Conclusions: CDK1/2 activity is a promising novel predictive marker to predict response to taxane- containing regimens for HER2- breast cancer. We could not predict taxane

sensitivity in HER2+ tumors; this finding is consistent with preclinical findings that HER2 overexpression is involved in abnormal SAC functionality. Thus, the functional SAC reflects taxane sensitivity in HER2- tumors. We are planning a large prospective study to validate these findings.

Session Info: General Poster Session, Saturday (2:00 PM-6:00 PM)Presentation Time: 02:00 PM Abstracts ASCO 2007

Abstracts ASCO 2007

[20521] Phase 2 dose multi-center, open-label study of ARQ 501, a checkpoint activator, in adult patients with persistent, recurrent or metastatic leiomyosarcoma (LMS).

L. P. Hartner, L. Rosen, M. Hensley, D. Mendelson, A. P. Staddon, W. Chow, O. Kovalyov, W. Ruka, K. Skladowski, A. Jagiello-Gruszfeld, M. Byakhov. University of Pennsylvania,

Philadelphia, PA, United States

Background: ARQ 501 selectively induces apoptosis in cancer cells by inducing a rapid and sustained increase of the pro-apoptotic protein E2F-1. ARQ 501 has been studied in three phase 1 studies,

demonstrating acceptable toxicity and encouraging signs of efficacy. A 54 y/o female with metastatic LMS who failed 7 previous therapies achieved a prolonged PR on ARQ 501 monotherapy. This was

consistent with preclinical data, where induction of E2F-1 and corresponding efficacy in human leiomyosarcoma xenografts was observed.

Methods: A phase 2 study in adult LMS patients (>3 prior systemic therapies) was initiated to assess ORR, TTP and further characterize the safety of ARQ 501. ORR included CR, PR and SD=4 mo. Four week cycles (ARQ 501 450mg/m2) were repeated until progression, unacceptable toxicity, or another

discontinuation criterion.Results: 49 patients were enrolled and 45 received ARQ 501. Data is available for 43 patients (4M/39F, median age, 54). Of the 43, 10 did not reach a protocol defined tumor assessment (4 deaths, 5 PD and 1 lost to follow-up prior to week 8), 19 have been assessed for response per RECIST at eight weeks (7 SD

of 8-28+ weeks, 1 PR, 11 PD) and 14 active patients yet to reach first tumor assessment.The most common AEs were: anemia (68%, 21%=G3), hyperbilirubinemia (35%, 6%=G3), fatigue (35%,

0%=G3), nausea (30%, 0%=G3), constipation (24%), hemolysis (21%, 6%=G3), dyspnea (21%), and vomiting (21%). One treatment related death was reported in a 47 y/o Asian male with severe hemolysis following a single infusion of ARQ 501 at 450 mg/m2. The pt was hospitalized, but severe hemolysis led

to acute renal failure and the patient expired after 4 days.Conclusions: ARQ 501 was administered to 45 patients with advanced, recurrent or persistent

leiomyosarcoma. Several patients have achieved some clinical benefit (1 PR, 3 prolonged SD), further analysis of efficacy data is warranted prior to additional clinical investigation.

Session Info: Published

Abstracts ASCO 2007

[5575] Cyclin-dependent kinase (CDK) activity as a prognostic marker in primary epithelial ovarian cancer.

J. A. de Souza, C. Wei, S. Noguchi, K. Lu, H. Ishihara, D. Aoki, N. T. Ueno. M. D. Anderson Cancer Center, Houston, TX, United States

Background: The unpredictable nature of primary epithelial ovarian cancer (EOC) response to treatment necessitates the identification of factors that predict long-term outcome. CDK activity is the core regulator of the cell-cycle checkpoint for cancer cells. We previously found that cell death induced

by taxanes requires increased activity of CDK1 and CDK2- fundamental kinases in cell proliferation. Thus, we hypothesized that CDK1/2 activities can determine the prognosis of patients with primary

EOC. Methods: We retrospectively analyzed frozen ovarian cancer specimens from 72 women treated with cytoreductive surgery and adjuvant chemotherapy for newly diagnosed EOC. Median patient age was 61 years (range, 38-80), 91% had FIGO stage III/IV disease, and 86% were treated with platinum- or taxane-based regimens. Median follow-up was 32 months; median time to treatment failure (TTF), defined as time from surgery to first relapse for patients who had achieved complete response or time

to disease progression for those who had persistent disease, was 15 months. Enzymatic activity of CDK1/2 in the tumor tissues was determined by a modified in vitro kinase activity assay using a C2P

device (Sysmex Inc.). Recursive partitioning and regression trees algorithm (RPART) was used to identify the cohorts based on CDK1/2 activities. Results: In the univariate analysis, disease stage (p=0.021), type of surgery (optimal vs. suboptimal) (p=0.004), presence of ascites (p=0.043), and

CA125 after the adjuvant therapy (p=0.005) were significant predictors of TTF. The cohort with CDK2 activity = 87.3 had a significantly shorter TTF (p=0.014) and the cohort with CDK1 activity = 226.35

showed a trend toward longer TTF (p=0.094). Multivariate Cox proportional hazards regression analysis revealed CDK2 activity (<87.3 vs. = 87.3) (p=0.012) and CA125 level at completion of the

adjuvant chemotherapy regimen (p=0.02) to be independent predictors of TTF. Conclusion: Our data supports kinase activity of cell cycle as a clinically significant prognostic factor. To extend these

findings, we plan to conduct a large prospective study to determine whether CDK1/2 profiling can predict long-term outcome in primary EOC.

Session Info: General Poster Session, Saturday (2:00 PM-6:00 PM)Presentation Time: 02:00 PM

Abstracts ASCO 2007

[18016] Gene expression levels of ribonucleotide reductase subunit M1 (RRM1), tyrosyl-DNA phosphodiesterase (Tdp1), nuclear factor of activated T-cells (NFAT),

and BubR1 mRNA expression in completely resected chemo-naive non-small cell lung cancer (NSCLC) patients.

I. Chaib, E. Jassem, M. Spkrzypski, R. Rosell, M. Taron, L. Perz-Roca, A. Szymanowska, W. Rzyman, G. Gulida, J. Jassem. ICO, Hospital Germans Trias i Pujol,

Badalona, Spain

Background: A relationship between gene transcripts and survival in operable NSCLC is now emerging. RRM1 is involved in DNA repair, Tdp1 is implicated in the repair of CPT-induced

topoisomerase damage, and NFAT promotes cancer invasion. BubR1 is a key spindle checkpoint gene, and altered BubR1 mRNA levels are associated with lymph node metastasis and chromosome instability. Methods: In order to identify p with a high risk of relapse, we examined the expression of these four genes in frozen resected tumors from 126 resected

NSCLC p by real-time quantitative PCR. Gene expression was normalized using ß-actin expression as internal reference. Results: Adenocarcinoma (adeno), 33 p; squamous cell carcinoma (SCC), 93 p. Stage: IA, 18 p; IB, 53 p; IIB, 33 p; IIIA, 22 p. Tumor transcript

expression: RRM1, 2.10; Tdp1, 1.77; NFAT, 0.56; BubR1, 16.40. Expression of RRM1, Tdp1 and BubR1 was higher in SCC than in adeno (P<0.001). Median time to relapse (TTR) was longer for p with low levels of RRM1 (P=0.11), Tdp1 (P=0.86), NFAT (P=0.29), or BubR1

(P=0.44) (Table). A significant trend towards longer survival was also observed in stage I p with low RRM1 P=0.06). In a multivariate Cox model, tumor size > 4 cm and stage III

predicted shorter TTR and survival. Conclusion: Increased mRNA expression of these genes is associated with shorter TTR; this knowledge could be useful for customizing adjuvant

chemotherapy.

Bibliografia

• Cancer Cell 2005, 8:7-12

• Cell Cycle 2005, 4(11):1495-99

• Cur Biol 2004, 11(24):R1001-04