RESUMO
Metabolic reprogramming is a common feature of many human cancers, including acute myeloid leukemia (AML). However, the upstream regulators that promote AML metabolic reprogramming and the benefits conferred to leukemia cells by these metabolic changes remain largely unknown. We report that the transcription factor ATF3 coordinates serine and nucleotide metabolism to maintain cell cycling, survival, and the differentiation blockade in AML. Analysis of mouse and human AML models demonstrate that ATF3 directly activates the transcription of genes encoding key enzymatic regulators of serine synthesis, one-carbon metabolism, and de novo purine and pyrimidine synthesis. Total steady-state polar metabolite and heavy isotope tracing analyses show that ATF3 inhibition reduces de novo serine synthesis, impedes the incorporation of serine-derived carbons into newly synthesized purines, and disrupts pyrimidine metabolism. Importantly, exogenous nucleotide supplementation mitigates the anti-leukemia effects of ATF3 inhibition. Together, these findings reveal the dependence of AML on ATF3-regulated serine and nucleotide metabolism.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Ciclo Celular , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleotídeos/metabolismo , Serina/metabolismo , Fator 3 Ativador da Transcrição/genética , Linhagem Celular Tumoral , Humanos , Leucemia Mieloide Aguda/genética , Proteínas de Neoplasias/genética , Nucleotídeos/genética , Serina/genéticaRESUMO
PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms â³11 and â³11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.
Assuntos
Proteína BRCA1 , Resistencia a Medicamentos Antineoplásicos , Éxons , Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Sítios de Splice de RNA , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Proteína BRCA1/genética , Feminino , Animais , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Mutação , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular TumoralRESUMO
The RNF168 E3 ubiquitin ligase is activated in response to double stranded DNA breaks (DSBs) where it mono-ubiquitinates γH2AX (ub-H2AX). RNF168 protein expression and ubiquitin signaling are finely regulated during the sensing, repair and resolution of DNA damage in order to avoid excessive spreading of ubiquitinated chromatin. Supra-physiological RNF168 protein expression levels have been shown to block DNA end resection at DSBs and increase PARP inhibitor (PARPi) sensitivity. In this study, we examined the impact of ectopic RNF168 overexpression on hydroxyurea (HU)-induced stalled replication forks in the setting of BRCA1 deficiency. Surprisingly, RNF168 overexpression resulted in the extension of DNA fibers, despite the presence of HU, in BRCA1 deficient cells. Mechanistically, RNF168 overexpression recruited RAD18 to ub-H2AX at HU-induced DNA breaks. Subsequently, a RAD18-SLF1 axis was responsible for initiating DNA synthesis in a manner that also required the break-induced replication (BIR) factors RAD52 and POLD3. Strikingly, the presence of wild-type BRCA1 blocked RNF168-induced DNA synthesis. Notably, BIR-like repair has previously been linked with tandem duplication events found in BRCA1-mutated genomes. Thus, in the absence of BRCA1, excessive RNF168 expression may drive BIR, and contribute to the mutational signatures observed in BRCA1-mutated cancers.
Assuntos
Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA/biossíntese , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Hidroxiureia/farmacologia , Reparo de DNA por Recombinação , UbiquitinaçãoRESUMO
The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.
Assuntos
Proteína BRCA1 , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína BRCA1/genética , Proteína BRCA2/genética , DNA/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Homóloga , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , HumanosRESUMO
The L-methioninase-annexin V/selenomethionine enzyme prodrug system, designed to target the tumor vasculature and release the methylselenol anticancer drug in the tumor, was tested in mice with implanted MBA-MB-231 breast tumors. This therapy was able to cause a reduction in the size of the tumors during the treatment period. It was shown that L-methioninase-annexin V was uniformly bound at the blood vessel surface in the tumor and also that there was a substantial cutoff of blood flowing through the treated tumor, consistent with the therapy's design. This new approach for enzyme prodrug therapy of breast cancer appears promising.
Assuntos
Anexina A5/metabolismo , Antineoplásicos/uso terapêutico , Liases de Carbono-Enxofre/metabolismo , Neoplasias Mamárias Animais/tratamento farmacológico , Metanol/análogos & derivados , Compostos Organosselênicos/uso terapêutico , Selenometionina/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Terapia Enzimática , Feminino , Humanos , Neoplasias Mamárias Animais/irrigação sanguínea , Metanol/uso terapêutico , Camundongos , Camundongos SCID , Transplante de Neoplasias , Pró-Fármacos/metabolismo , Pró-Fármacos/uso terapêuticoRESUMO
This paper focuses on the targeting of single-walled carbon nanotubes (SWNTs) for the treatment of breast cancer with minimal side effects using photothermal therapy. The human protein annexin V (AV) binds specifically to anionic phospholipids expressed externally on the surface of tumour cells and endothelial cells that line the tumour vasculature. A 2 h incubation of the SWNT-AV conjugate with proliferating endothelial cells followed by washing and near-infrared (NIR) irradiation at a wavelength of 980 nm was enough to induce significant cell death; there was no significant cell death with irradiation or the conjugate alone. Administration of the same conjugate i.v. in BALB/c female mice with implanted 4T1 murine mammary at a dose of 0.8 mg SWNT kg(-1) and followed one day later by NIR irradiation of the tumour at a wavelength of 980 nm led to complete disappearance of implanted 4T1 mouse mammary tumours for the majority of the animals by 11 days since the irradiation. The combination of the photothermal therapy with the immunoadjuvant cyclophosphamide resulted in increased survival. The in vivo results suggest the SWNT-AV/NIR treatment is a promising approach to treat breast cancer.
Assuntos
Hipertermia Induzida , Neoplasias Mamárias Animais/terapia , Nanotubos de Carbono/química , Fototerapia , Animais , Anexina A5/isolamento & purificação , Anexina A5/metabolismo , Biotinilação , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Maleimidas/química , Neoplasias Mamárias Animais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Força Atômica , Microscopia de Fluorescência , Fosfatidiletanolaminas/química , Polietilenoglicóis/química , Proteínas Recombinantes/isolamento & purificação , Espectroscopia de Luz Próxima ao Infravermelho , Coloração e Rotulagem , SuspensõesRESUMO
Homologous recombination (HR)-deficiency induces a dependency on DNA polymerase theta (Polθ/Polq)-mediated end joining, and Polθ inhibitors (Polθi) are in development for cancer therapy. BRCA1 and BRCA2 deficient cells are thought to be synthetic lethal with Polθ, but whether distinct HR gene mutations give rise to equivalent Polθ-dependence, and the events that drive lethality, are unclear. In this study, we utilized mouse models with separate Brca1 functional defects to mechanistically define Brca1-Polθ synthetic lethality. Surprisingly, homozygous Brca1 mutant, Polq-/- cells were viable, but grew slowly and had chromosomal instability. Brca1 mutant cells proficient in DNA end resection were significantly more dependent on Polθ for viability; here, treatment with Polθi elevated RPA foci, which persisted through mitosis. In an isogenic system, BRCA1 null cells were defective, but PALB2 and BRCA2 mutant cells exhibited active resection, and consequently stronger sensitivity to Polθi. Thus, DNA end resection is a critical determinant of Polθi sensitivity in HR-deficient cells, and should be considered when selecting patients for clinical studies.
Assuntos
Proteína BRCA1 , Genes BRCA2 , Camundongos , Animais , Humanos , Proteína BRCA1/genética , Mutação , Mutações Sintéticas Letais , DNARESUMO
Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.
RESUMO
BRCA1 splice isoforms Δ11 and Δ11q can contribute to PARP inhibitor (PARPi) resistance by splicing-out the mutation-containing exon, producing truncated, partially-functional proteins. However, the clinical impact and underlying drivers of BRCA1 exon skipping remain undetermined. We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs), predicted in silico to drive exon skipping. Predictions were confirmed using qRT-PCR, RNA sequencing, western blots and BRCA1 minigene modelling. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials. We demonstrate that SSMs drive BRCA1 exon 11 skipping and PARPi resistance, and should be clinically monitored, along with frame-restoring secondary mutations.
RESUMO
DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.
Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína BRCA1/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/genética , DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Ligação Proteica , Transporte Proteico , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes BRCA1 and BRCA2 (BRCA). Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. SIGNIFICANCE: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype "BRCAness," yielding promising biomarkers, targets, and opportunities to resensitize refractory disease.See related commentary by Canman, p. 1214.
Assuntos
Proteína BRCA2 , Replicação do DNA , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Genes BRCA2 , Recombinação Homóloga , HumanosRESUMO
Cancers that arise from BRCA1 germline mutations are deficient for homologous recombination (HR) DNA repair and are sensitive to DNA-damaging agents such as platinum and PARP inhibitors. In vertebrate organisms, knockout of critical HR genes including BRCA1 and BRCA2 is lethal because HR is required for genome replication. Thus, cancers must develop strategies to cope with loss of HR activity. Furthermore, as established tumors respond to chemotherapy selection pressure, additional genetic adaptations transition cancers to an HR-proficient state. In this review, we discuss biological mechanisms that influence the ability of BRCA1-mutant cancers to perform HR. Furthermore, we consider how the HR status fluctuates throughout the cancer life course, from tumor initiation to the development of therapy refractory disease.
Assuntos
Proteína BRCA1/genética , Carcinogênese/genética , Reparo de DNA por Recombinação/genética , Animais , Mutação em Linhagem Germinativa , HumanosRESUMO
BRCA1 gene mutations impair homologous recombination (HR) DNA repair, resulting in cellular senescence and embryonic lethality in mice. Therefore, BRCA1-deficient cancers require adaptations that prevent excessive genomic alterations from triggering cell death. RNF168-mediated ubiquitination of γH2AX at K13/15 (ub-H2AX) serves as a recruitment module for the localization of 53BP1 to DNA break sites. Here, we found multiple BRCA1-mutant cancer cell lines and primary tumors with low levels of RNF168 protein expression. Overexpression of ectopic RNF168 or a ub-H2AX fusion protein induced cell death and delayed BRCA1-mutant tumor formation. Cell death resulted from the recruitment of 53BP1 to DNA break sites and inhibition of DNA end resection. Strikingly, reintroduction of BRCA1 or 53BP1 depletion restored HR and rescued the ability of cells to maintain RNF168 and ub-H2AX overexpression. Thus, downregulation of RNF168 protein expression is a mechanism for providing BRCA1-null cancer cell lines with a residual level of HR that is essential for viability. Overall, our work identifies loss of RNF168 ubiquitin signaling as a proteomic alteration that supports BRCA1-mutant carcinogenesis. We propose that restoring RNF168-ub-H2AX signaling, potentially through inhibition of deubiquitinases, could represent a new therapeutic approach. SIGNIFICANCE: This study explores the concept that homologous recombination DNA repair is not an all-or-nothing concept, but a spectrum, and that where a tumor stands on this spectrum may have therapeutic relevance.See related commentary by Wang and Wulf, p. 2720.
Assuntos
Neoplasias , Ubiquitina , Animais , Proteína BRCA1/genética , Dano ao DNA , Camundongos , Neoplasias/genética , Proteômica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Pharmacological inhibition of PARP is a promising approach in treating high grade serous ovarian carcinoma (HGSOC). PARP inhibitors (PARPi) are most active in patients with defects in DNA damage repair (DDR) mechanisms, such as alterations in expression/function of DNA repair and homologous recombination (HR) genes/proteins, including BRCA1 and BRCA2. Benefit of PARPi could be extended towards HR-proficient patients by combining PARPi with agents that functionally abrogate HR. An attractive molecular target for this purpose is heat shock protein 90 (HSP90), which mediates the maturation and stability of several key proteins required for DDR. Here, we tested the hypothesis that targeted inhibition of HSP90 with a small-molecule inhibitor ganetespib would sensitize non-BRCA mutant ovarian carcinoma (OC) cells to PARP inhibition by talazoparib. We used commercially available cell lines, along with several novel HGSOC OC cell lines established in our laboratory. Ganetespib treatment destabilized HSP90 client proteins involved in DNA damage response and cell cycle checkpoint, and disrupted γ-irradiation-induced DNA repair. The effects of the combination of ganetespib and talazoparib on OC cell viability and survival were also analyzed, and among the non-BRCA mutant cell lines analyzed, the combination was synergistic in several cell lines (OVCAR-3, OC-1, OC-16). Together, our data suggest that ganetespib-mediated inhibition of HSP90 effectively disrupts critical DDR pathway proteins and may sensitize OC cells without 'BRCAness' to PARPi. From a clinical perspective, this suggests that HSP90 inhibition has the potential to sensitize some HGSOC patients without HR pathway alterations to PARPi, and potentially other DNA-damage inducing agents.
Assuntos
Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Proteínas de Choque Térmico HSP90/genética , Recombinação Homóloga , Humanos , Imuno-Histoquímica , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Ftalazinas/farmacologia , Radiação Ionizante , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BRCA1 mutant carcinomas are sensitive to PARP inhibitor (PARPi) therapy; however, resistance arises. BRCA1 BRCT domain mutant proteins do not fold correctly and are subject to proteasomal degradation, resulting in PARPi sensitivity. In this study, we show that cell lines and patient-derived tumors, with highly disruptive BRCT domain mutations, have readily detectable BRCA1 protein expression, and are able to proliferate in the presence of PARPi. Peptide analyses reveal that chemo-resistant cancers contain residues encoded by BRCA1 intron 15. Mechanistically, cancers with BRCT domain mutations harbor BRCA1 gene breakpoints within or adjacent to Alu elements in intron 15; producing partial gene duplications, inversions and translocations, and terminating transcription prior to the mutation-containing BRCT domain. BRCA1 BRCT domain-deficient protein isoforms avoid mutation-induced proteasomal degradation, support homology-dependent DNA repair, and promote PARPi resistance. Taken together, Alu-mediated BRCA1 gene rearrangements are responsible for generating hypomorphic proteins, and may represent a biomarker of PARPi resistance.
Assuntos
Elementos Alu , Antineoplásicos/administração & dosagem , Proteína BRCA1/genética , Neoplasias da Mama/genética , Rearranjo Gênico , Íntrons , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Animais , Proteína BRCA1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Inversão Cromossômica , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Camundongos Nus , Translocação GenéticaRESUMO
BRCA1 functions in homologous recombination (HR) both up- and downstream of DNA end resection. However, in cells with 53BP1 gene knockout (KO), BRCA1 is dispensable for the initiation of resection, but whether BRCA1 activity is entirely redundant after end resection is unclear. Here, we found that 53bp1 KO rescued the embryonic viability of a Brca1ΔC/ΔC mouse model that harbors a stop codon in the coiled-coil domain. However, Brca1ΔC/ΔC;53bp1-/- mice were susceptible to tumor formation, lacked Rad51 foci, and were sensitive to PARP inhibitor (PARPi) treatment, indicative of suboptimal HR. Furthermore, BRCA1 mutant cancer cell lines were dependent on truncated BRCA1 proteins that retained the ability to interact with PALB2 for 53BP1 KO induced RAD51 foci and PARPi resistance. Our data suggest that the overall efficiency of 53BP1 loss of function induced HR may be BRCA1 mutation dependent. In the setting of 53BP1 KO, hypomorphic BRCA1 proteins are active downstream of end resection, promoting RAD51 loading and PARPi resistance.
Assuntos
Proteína BRCA1/genética , Resistencia a Medicamentos Antineoplásicos , Recombinação Homóloga , Neoplasias Mamárias Experimentais/genética , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína BRCA1/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Feminino , Células HEK293 , Humanos , Mutação com Perda de Função , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismoRESUMO
Accurately identifying patients with high-grade serous ovarian carcinoma (HGSOC) who respond to poly(ADP-ribose) polymerase inhibitor (PARPi) therapy is of great clinical importance. Here we show that quantitative BRCA1 methylation analysis provides new insight into PARPi response in preclinical models and ovarian cancer patients. The response of 12 HGSOC patient-derived xenografts (PDX) to the PARPi rucaparib was assessed, with variable dose-dependent responses observed in chemo-naive BRCA1/2-mutated PDX, and no responses in PDX lacking DNA repair pathway defects. Among BRCA1-methylated PDX, silencing of all BRCA1 copies predicts rucaparib response, whilst heterozygous methylation is associated with resistance. Analysis of 21 BRCA1-methylated platinum-sensitive recurrent HGSOC (ARIEL2 Part 1 trial) confirmed that homozygous or hemizygous BRCA1 methylation predicts rucaparib clinical response, and that methylation loss can occur after exposure to chemotherapy. Accordingly, quantitative BRCA1 methylation analysis in a pre-treatment biopsy could allow identification of patients most likely to benefit, and facilitate tailoring of PARPi therapy.
Assuntos
Proteína BRCA1/genética , Metilação de DNA , Indóis/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/farmacologia , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Dosagem de Genes , Humanos , Estimativa de Kaplan-Meier , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Células Tumorais CultivadasRESUMO
Mutant cystathionine gamma-lyase was targeted to phosphatidylserine exposed on tumor vasculature through fusion with Annexin A1 or Annexin A5. Cystathionine gamma-lyase E58N, R118L, and E338N mutations impart nonnative methionine gamma-lyase activity, resulting in tumor-localized generation of highly toxic methylselenol upon systemic administration of nontoxic selenomethionine. The described therapeutic system circumvents systemic toxicity issues using a novel drug delivery/generation approach and avoids the administration of nonnative proteins and/or DNA required with other enzyme prodrug systems. The enzyme fusion exhibits strong and stable in vitro binding with dissociation constants in the nanomolar range for both human and mouse breast cancer cells and in a cell model of tumor vascular endothelium. Daily administration of the therapy suppressed growth of highly aggressive triple-negative murine 4T1 mammary tumors in immunocompetent BALB/cJ mice and MDA-MB-231 tumors in SCID mice. Treatment did not result in the occurrence of negative side effects or the elicitation of neutralizing antibodies. On the basis of the vasculature-targeted nature of the therapy, combinations with rapamycin and cyclophosphamide were evaluated. Rapamycin, an mTOR inhibitor, reduces the prosurvival signaling of cells in a hypoxic environment potentially exacerbated by a vasculature-targeted therapy. IHC revealed, unsurprisingly, a significant hypoxic response (increase in hypoxia-inducible factor 1 α subunit, HIF1A) in the enzyme prodrug-treated tumors and a dramatic reduction of HIF1A upon rapamycin treatment. Cyclophosphamide, an immunomodulator at low doses, was combined with the enzyme prodrug therapy and rapamycin; this combination synergistically reduced tumor volumes, inhibited metastatic progression, and enhanced survival. Mol Cancer Ther; 16(9); 1855-65. ©2017 AACR.
Assuntos
Ciclofosfamida/farmacologia , Neoplasias/enzimologia , Neoplasias/patologia , Neovascularização Patológica/enzimologia , Pró-Fármacos/farmacologia , Sirolimo/farmacologia , Animais , Anexina A5/genética , Liases de Carbono-Enxofre/genética , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neovascularização Patológica/tratamento farmacológico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Patients with cancers that harbor breast cancer 1 (BRCA1) mutations initially respond well to platinum and poly(ADP-ribose) polymerase inhibitor (PARPi) therapy; however, resistance invariably arises in these patients and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site that is thought to produce a shortened, nonfunctional peptide. In this study, we investigated the mechanisms that lead to PARPi and platinum resistance in the SUM1315MO2 breast cancer cell line, which harbors a hemizygous BRCA1185delAG mutation. SUM1315MO2 cells were initially sensitive to PARPi and cisplatin but readily acquired resistance. PARPi- and cisplatin-resistant clones did not harbor secondary reversion mutations; rather, PARPi and platinum resistance required increased expression of a really interesting gene (RING) domain-deficient BRCA1 protein (Rdd-BRCA1). Initiation of translation occurred downstream of the frameshift mutation, probably at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BRCA1-associated RING domain 1 (BARD1) interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance. Furthermore, Rdd-BRCA1 protein expression was detected in recurrent carcinomas from patients who carried germline BRCA1185delAG mutations. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic and capable of contributing to PARPi and platinum resistance when expressed at high levels.
Assuntos
Proteína BRCA1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Platina/farmacologia , Animais , Anticorpos Monoclonais/química , Neoplasias da Mama/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cisplatino/farmacologia , Éxons , Feminino , Mutação em Linhagem Germinativa , Humanos , Camundongos , Camundongos Endogâmicos NOD , Mutação , Transplante de Neoplasias , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Domínios ProteicosRESUMO
Breast and ovarian cancer patients harboring BRCA1/2 germline mutations have clinically benefitted from therapy with PARP inhibitor (PARPi) or platinum compounds, but acquired resistance limits clinical impact. In this study, we investigated the impact of mutations on BRCA1 isoform expression and therapeutic response. Cancer cell lines and tumors harboring mutations in exon 11 of BRCA1 express a BRCA1-Δ11q splice variant lacking the majority of exon 11. The introduction of frameshift mutations to exon 11 resulted in nonsense-mediated mRNA decay of full-length, but not the BRCA1-Δ11q isoform. CRISPR/Cas9 gene editing as well as overexpression experiments revealed that the BRCA1-Δ11q protein was capable of promoting partial PARPi and cisplatin resistance relative to full-length BRCA1, both in vitro and in vivo Furthermore, spliceosome inhibitors reduced BRCA1-Δ11q levels and sensitized cells carrying exon 11 mutations to PARPi treatment. Taken together, our results provided evidence that cancer cells employ a strategy to remove deleterious germline BRCA1 mutations through alternative mRNA splicing, giving rise to isoforms that retain residual activity and contribute to therapeutic resistance. Cancer Res; 76(9); 2778-90. ©2016 AACR.