RESUMO
The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).
Assuntos
Leucemia Mieloide Aguda , Células-Tronco Neoplásicas , Linfócitos T , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/patologia , Animais , Camundongos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Subunidade alfa de Receptor de Interleucina-3/imunologia , Subunidade alfa de Receptor de Interleucina-3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Citotoxicidade ImunológicaRESUMO
PURPOSE: To assess the usefulness of combining hyperthermia with a DNA repair inhibitor (double-strand break bait [Dbait]) and its potential application to radiofrequency ablation (RFA) in a preclinical model of human colorectal cancer. MATERIALS AND METHODS: The local ethics committee of animal experimentation approved all investigations. First, the relevance was assessed by studying the survival of four human colorectal adenocarcinoma cell cultures after 1 hour of hyperthermia at 41°C or 43°C with or without Dbait. Human colon adenocarcinoma cells (HT-29) were grafted subcutaneously into nude mice (n = 111). When tumors reached approximately 500 mm(3), mice were treated with Dbait alone (n = 20), sublethal RFA (n = 21), three different Dbait schemes and sublethal RFA (n = 52), or a sham treatment (n = 18). RFA was performed to ablate the tumor center alone. To elucidate antitumor mechanisms, 39 mice were sacrificed for blinded pathologic analysis, including assessment of DNA damage, cell proliferation, and tumor necrosis. Others were monitored for tumor growth and survival. Analyses of variance and log-rank tests were used to evaluate differences. RESULTS: When associated with mild hyperthermia, Dbait induced cytotoxicity in all tested colon cancer cell lines. Sublethal RFA or Dbait treatment alone moderately improved survival (median, 40 days vs 28 days for control; P = .0005) but combination treatment significantly improved survival (median, 84 days vs 40 days for RFA alone, P = .0004), with approximately half of the animals showing complete tumor responses. Pathologic studies showed that the Dbait and RFA combination strongly enhances DNA damage and coagulation areas in tumors. CONCLUSION: Combining Dbait with RFA sensitizes the tumor periphery to mild hyperthermia and increases RFA antitumor efficacy.
Assuntos
Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/cirurgia , Ablação por Cateter , Neoplasias Colorretais/patologia , Reparo do DNA/efeitos dos fármacos , Hipertermia Induzida , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Oligodesoxirribonucleotídeos/farmacologia , Adenocarcinoma/patologia , Animais , Dano ao DNA/efeitos dos fármacos , Humanos , Camundongos , Células Tumorais CultivadasRESUMO
One of the major early steps of repair is the recruitment of repair proteins at the damage site, and this is coordinated by a cascade of modifications controlled by phosphatidylinositol 3-kinase-related kinases and/or poly (ADP-ribose) polymerase (PARP). We used short interfering DNA molecules mimicking double-strand breaks (called Dbait) or single-strand breaks (called Pbait) to promote DNA-dependent protein kinase (DNA-PK) and PARP activation. Dbait bound and induced both PARP and DNA-PK activities, whereas Pbait acts only on PARP. Therefore, comparative study of the two molecules allows analysis of the respective roles of the two signaling pathways: both recruit proteins involved in single-strand break repair (PARP, XRCC1 and PCNA) and prevent their recruitment at chromosomal damage. Dbait, but not Pbait, also inhibits recruitment of proteins involved in double-strand break repair (53BP1, NBS1, RAD51 and DNA-PK). By these ways, Pbait and Dbait disorganize DNA repair, thereby sensitizing cells to various treatments. Single-strand breaks repair inhibition depends on direct trapping of the main proteins on both molecules. Double-strand breaks repair inhibition may be indirect, resulting from the phosphorylation of double-strand breaks repair proteins and chromatin targets by activated DNA-PK. The DNA repair inhibition by both molecules is confirmed by their synthetic lethality with BRCA mutations.
Assuntos
Dano ao DNA , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Transdução de Sinais , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Sequência de Bases , Benzimidazóis/farmacologia , Quebras de DNA de Cadeia Dupla , Proteína Quinase Ativada por DNA/genética , Ativação Enzimática , Genoma Humano , Células HeLa , Humanos , Proteínas Nucleares/genética , Fosforilação , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/genéticaRESUMO
BACKGROUND: The hallmark of HIV-1 pathogenesis is the progressive CD4(+) T cell depletion and high propensity of CD4(+) T cells to apoptosis. HIV-1 viral protein R (Vpr) is a major pro-apoptotic gene product. A first Vpr-mediated apoptotic mechanism that requires a physical interaction of HIV-1 Vpr(71-82) mitochondriotoxic domain containing the conserved sequence (71-)HFRIGCRHSRIG(-82) with the Adenine Nucleotide Translocator (ANT) has been characterized. The family of Ser/Thr protein phosphatase PP2A interacts with several viral proteins to regulate cell growth and apoptotic pathways. Previous studies based on yeast two hybrid assays and mutational experiments indicated that PP2A(1) is involved in the induction of G2 arrest by HIV-1 Vpr. PRINCIPAL FINDINGS: Experiments combining pull-down, cell penetration and apoptosis analyses in distinct human cells indicate that the PP2A(1) binding sequence from Vpr(77-92) is a new cell penetrating apoptotic sequence. We also found that the I84P mutation or the IIQ/VTR(83-85) and T89A substitutions in the Vpr(77-92) sequence prevent PP2A(1) binding, cell penetration and apoptosis. In addition the double R77A and R80A mutation known to inactivate the mitochondriotoxic Vpr(71-82) domain, has no effect on the biological properties of the Vpr(77-92) domain. CONCLUSION: Together our data provide evidence for the first time that the Vpr(77-92) sequence delineates a biological active domain of Vpr with PP2A(1) binding and pro-apoptotic capacities and, it is conceivable that this cell penetrating sequence may account for the Vpr internalization in uninfected cells. Finally, our data also implicate the existence of two partially overlapping pro-apoptotic domains in the Vpr C-terminal part, a redundancy that represents a new approach to address the question of biological relevance of HIV-1 Vpr. In this context, future studies will be required to determine the functional relevance of the Vpr(77-92) domain in full length Vpr protein and also in entire HIV provirus.
Assuntos
Apoptose/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteína Fosfatase 2/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Biotinilação , Linhagem Celular Tumoral , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Células Jurkat , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/genética , Ligação Proteica , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/química , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genéticaRESUMO
BACKGROUND: Previous studies established that PP1 is a target for Bcl-2 proteins and an important regulator of apoptosis. The two distinct functional PP1 consensus docking motifs, R/Kx((0,1))V/IxF and FxxR/KxR/K, involved in PP1 binding and cell death were previously characterized in the BH1 and BH3 domains of some Bcl-2 proteins. PRINCIPAL FINDINGS: In this study, we demonstrate that DPT-AIF(1), a peptide containing the AIF(562-571) sequence located in a c-terminal domain of AIF, is a new PP1 interacting and cell penetrating molecule. We also showed that DPT-AIF(1) provoked apoptosis in several human cell lines. Furthermore, DPT-APAF(1) a bi-partite cell penetrating peptide containing APAF-1(122-131), a non penetrating sequence from APAF-1 protein, linked to our previously described DPT-sh1 peptide shuttle, is also a PP1-interacting death molecule. Both AIF(562-571) and APAF-1(122-131) sequences contain a common R/Kx((0,1))V/IxFxxR/KxR/K motif, shared by several proteins involved in control of cell survival pathways. This motif combines the two distinct PP1c consensus docking motifs initially identified in some Bcl-2 proteins. Interestingly DPT-AIF(2) and DPT-APAF(2) that carry a F to A mutation within this combinatorial motif, no longer exhibited any PP1c binding or apoptotic effects. Moreover the F to A mutation in DPT-AIF(2) also suppressed cell penetration. CONCLUSION: These results indicate that the combinatorial PP1c docking motif R/Kx((0,1))V/IxFxxR/KxR/K, deduced from AIF(562-571) and APAF-1(122-131) sequences, is a new PP1c-dependent Apoptotic Signature. This motif is also a new tool for drug design that could be used to characterize potential anti-tumour molecules.