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1.
Mol Cancer Ther ; 21(7): 1090-1102, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35439320

RESUMO

Exatecan and deruxtecan are antineoplastic camptothecin derivatives in development as tumor-targeted-delivery warheads in various formulations including peptides, liposomes, polyethylene glycol nanoparticles, and antibody-drug conjugates. Here, we report the molecular pharmacology of exatecan compared with the clinically approved topoisomerase I (TOP1) inhibitors and preclinical models for validating biomarkers and the combination of exatecan with ataxia telangiectasia and Rad3-related kinase (ATR) inhibitors. Modeling exatecan binding at the interface of a TOP1 cleavage complex suggests two novel molecular interactions with the flanking DNA base and the TOP1 residue N352, in addition to the three known interactions of camptothecins with the TOP1 residues R364, D533, and N722. Accordingly, exatecan showed much stronger TOP1 trapping, higher DNA damage, and apoptotic cell death than the classical TOP1 inhibitors used clinically. We demonstrate the value of SLFN11 expression and homologous recombination (HR) deficiency (HRD) as predictive biomarkers of response to exatecan. We also show that exatecan kills cancer cells synergistically with the clinical ATR inhibitor ceralasertib (AZD6738). To establish the translational potential of this combination, we tested CBX-12, a clinically developed pH-sensitive peptide-exatecan conjugate that selectively targets cancer cells and is currently in clinical trials. The combination of CBX-12 with ceralasertib significantly suppressed tumor growth in mouse xenografts. Collectively, our results demonstrate the potency of exatecan as a TOP1 inhibitor and its clinical potential in combination with ATR inhibitors, using SLFN11 and HRD as predictive biomarkers.


Assuntos
DNA Topoisomerases Tipo I , Neoplasias , Inibidores da Topoisomerase I , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Camptotecina/análogos & derivados , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Humanos , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Nucleares/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores da Topoisomerase I/farmacologia
2.
Immunotherapy ; 14(18): 1467-1480, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36597724

RESUMO

Aims: To determine whether antigen-independent targeting of the TOP1 inhibitor exatecan to tumor with a pH-sensitive peptide (CBX-12) produces superior synergy with immunotherapy compared with unconjugated exatecan. Materials & methods: In vitro and ex vivo functional assays were performed via FACS and ELISA assays. In vivo efficacy was evaluated in the syngeneic CT26 model. Results: CBX-12 combined with anti-PD-1 or anti-CTLA4 results in delayed tumor growth and complete response, with cured animals displaying long-term antitumor immunity. CBX-12 stimulates expression of MHC 1 and PD-L1 and is an inducer of immunogenic cell death, producing long-term immune recognition of tumor cells and resultant antitumor immunity. Conclusion: The authors' data provide the rationale for exploring immunotherapy combinations with CBX-12 in clinical trials.


Although combinations of chemotherapy and immunotherapy have shown great promise for cancer treatment, they have also demonstrated significant safety concerns that require dose reductions. Targeting chemotherapy to the tumor can avoid these safety issues, thereby enhancing efficacy of combination therapies. CBX-12 is a novel peptide-drug agent targeting the TOP1-inhibiting drug exatecan to tumor via pH-sensitive peptide. Unlike tumor targeting via antibody, CBX-12 universally targets all solid tumors. CBX-12 avoids the immune cell toxicity of nontumor-targeted exatecan and safely synergizes with immunotherapies. CBX-12 treatment causes tumor cells to express and secrete molecules that result in activation of immune components to recognize and eliminate tumor cells. These data support the upcoming clinical trials of CBX-12 in combination with immunotherapy.


Assuntos
Neoplasias , Animais , Neoplasias/tratamento farmacológico , Camptotecina/uso terapêutico , Imunoterapia/métodos , Imunidade , Linhagem Celular Tumoral , Microambiente Tumoral
4.
NAR Cancer ; 3(2): zcab021, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34316708

RESUMO

Topoisomerase inhibitors are potent DNA damaging agents which are widely used in oncology, and they demonstrate robust synergistic tumor cell killing in combination with DNA repair inhibitors, including poly(ADP)-ribose polymerase (PARP) inhibitors. However, their use has been severely limited by the inability to achieve a favorable therapeutic index due to severe systemic toxicities. Antibody-drug conjugates address this issue via antigen-dependent targeting and delivery of their payloads, but this approach requires specific antigens and yet still suffers from off-target toxicities. There is a high unmet need for a more universal tumor targeting technology to broaden the application of cytotoxic payloads. Acidification of the extracellular milieu arises from metabolic adaptions associated with the Warburg effect in cancer. Here we report the development of a pH-sensitive peptide-drug conjugate to deliver the topoisomerase inhibitor, exatecan, selectively to tumors in an antigen-independent manner. Using this approach, we demonstrate potent in vivo cytotoxicity, complete suppression of tumor growth across multiple human tumor models, and synergistic interactions with a PARP inhibitor. These data highlight the identification of a peptide-topoisomerase inhibitor conjugate for cancer therapy that provides a high therapeutic index, and is applicable to all types of human solid tumors in an antigen-independent manner.

5.
Blood Adv ; 3(22): 3661-3673, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31751472

RESUMO

Acute myeloid leukemias (AML) harboring a constitutively active internal tandem duplication (ITD) mutation in the FMS-like kinase tyrosine kinase (FLT3) receptor are associated with poor patient prognosis. Despite initial clinical responses to FLT3 kinase inhibitors, patients eventually relapse. Mechanisms of resistance include the acquisition of secondary FLT3 mutations and protective stromal signaling within the bone marrow niche. Here we show that LAM-003, a prodrug of the heat shock protein 90 inhibitor LAM-003A, has cytotoxic activity against AML cell lines and primary samples harboring FLT3-ITD. LAM-003 regressed tumors in an MV-4-11 xenograft mouse model and extended survival in a MOLM-13 systemic model. LAM-003 displayed synergistic activity with chemotherapeutic drugs and FLT3 inhibitors, with the most robust synergy being obtained with venetoclax, a BCL-2 inhibitor. This finding was verified in a MOLM-13 systemic survival model in which the combination significantly prolonged survival compared with the single agents. Importantly, LAM-003 exhibited equipotent activity against FLT3 inhibitor-resistant mutants of FLT3, such as D835 or F691, in cytotoxic and FLT3 degradation assays. LAM-003 also retained potency in AML cells grown in stromal-conditioned media that were resistant to FLT3 inhibitors. Lastly, a genome-wide CRISPR screen revealed epigenetic regulators, including KDM6A, as determinants of LAM-003 sensitivity in AML cell lines, leading to the discovery of synergy with an EZH2 inhibitor. Collectively, these preclinical findings support the use of LAM-003 in FLT3-ITD patients with AML who no longer respond to FLT3 inhibitor therapy either as a single agent or in combination with drugs known to be active in AML.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Duplicação Gênica , Leucemia Mieloide Aguda/genética , Inibidores de Proteínas Quinases/farmacologia , Tirosina Quinase 3 Semelhante a fms/genética , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Epigênese Genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Camundongos , Mutação , Inibidores de Proteínas Quinases/uso terapêutico
6.
Autophagy ; 13(6): 1082-1083, 2017 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-28350209

RESUMO

We identified the PIKFYVE inhibitor apilimod as a potent and selective cytotoxic agent against B-cell non-Hodgkin lymphoma (B-NHL). Our data robustly establish PIKFYVE as the target through which apilimod kills B-NHL cells and show that apilimod-induced death in B-NHL is mediated by broad disruption of lysosome homeostasis characterized by lysosomal swelling, TFEB nuclear translocation, impaired maturation of lysosomal enzymes and incomplete autophagosome clearance. Furthermore, through genome-wide CRISPR knockout screening, we identified specific lysosomal genes (TFEB, CLCN7, OSTM1 and SNX10) as critical determinants of apilimod-induced cytotoxicity. Together these data highlight disruption of lysosome homeostasis through PIKFYVE inhibition as a novel anticancer mechanism in B-NHL and potentially other cancers.


Assuntos
Linfócitos B/patologia , Linfoma não Hodgkin/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Endossomos/metabolismo , Humanos , Linfoma não Hodgkin/enzimologia , Linfoma não Hodgkin/patologia , Lisossomos/metabolismo , Modelos Biológicos , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia
7.
Blood ; 129(13): 1768-1778, 2017 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-28104689

RESUMO

We identified apilimod as an antiproliferative compound by high-throughput screening of clinical-stage drugs. Apilimod exhibits exquisite specificity for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) lipid kinase and has selective cytotoxic activity in B-cell non-Hodgkin lymphoma (B-NHL) compared with normal cells. Apilimod displays nanomolar activity in vitro, and in vivo studies demonstrate single-agent efficacy as well as synergy with approved B-NHL drugs. Using biochemical and knockdown approaches, and discovery of a kinase domain mutation conferring resistance, we demonstrate that apilimod-mediated cytotoxicity is driven by PIKfyve inhibition. Furthermore, a critical role for lysosome dysfunction as a major factor contributing to apilimod's cytotoxicity is supported by a genome-wide CRISPR screen. In the screen, TFEB (master transcriptional regulator of lysosomal biogenesis) and endosomal/lysosomal genes CLCN7, OSTM1, and SNX10 were identified as important determinants of apilimod sensitivity. These findings thus suggest that disruption of lysosomal homeostasis with apilimod represents a novel approach to treat B-NHL.


Assuntos
Linfoma de Células B/tratamento farmacológico , Morfolinas/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/uso terapêutico , Triazinas/uso terapêutico , Antineoplásicos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Avaliação Pré-Clínica de Medicamentos/métodos , Endossomos/efeitos dos fármacos , Endossomos/genética , Ensaios de Triagem em Larga Escala , Humanos , Hidrazonas , Lisossomos/efeitos dos fármacos , Lisossomos/genética , Fosfatidilinositol 3-Quinases , Pirimidinas
8.
Stem Cell Reports ; 4(5): 926-38, 2015 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-25866159

RESUMO

The mechanisms regulating human embryonic stem (ES) cell self-renewal and differentiation are not well defined in part due to the lack of tools for forward genetic analysis. We present a piggyBac transposon gain of function screen in human ES cells that identifies DENND2C, which genetically cooperates with NANOG to maintain self-renewal in the presence of retinoic acid. We show that DENND2C negatively regulates RHOA activity, which cooperates with NANOG to block differentiation. It has been recently shown that RHOA exists in the nucleus and is activated by DNA damage; however, its nuclear function remains unknown. We discovered that RHOA associates with DNA and that DENND2C affects nuclear RHOA localization, activity, and DNA association. Our study illustrates the power of piggyBac as a cost-effective, efficient, and easy to use tool for forward genetic screens in human ES cells and provides insight into the role of RHOA in the nucleus.


Assuntos
Elementos de DNA Transponíveis/genética , Vetores Genéticos/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Mapeamento Cromossômico , Vetores Genéticos/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Mutagênese Insercional , Proteína Homeobox Nanog , Tretinoína/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismo
9.
Genetics ; 189(1): 123-35, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21705760

RESUMO

Microtubules are dynamic structures that must elongate, disassemble, and be cleaved into smaller pieces for proper neuronal development and function. The AAA ATPase Spastin severs microtubules along their lengths and is thought to regulate the balance between long, stable filaments and shorter fragments that seed extension or are transported. In both Drosophila and humans, loss of Spastin function results in reduction of synaptic connections and disabling motor defects. To gain insight into how spastin is regulated, we screened the Drosophila melanogaster genome for deletions that modify a spastin overexpression phenotype, eye size reduction. One suppressor region deleted p21-activated kinase 3 (pak3), which encodes a member of the Pak family of actin-regulatory enzymes, but whose in vivo function is unknown. We show that pak3 mutants have only mild synaptic defects at the larval neuromuscular junction, but exhibit a potent genetic interaction with spastin mutations. Aberrant bouton morphology, microtubule distribution, and synaptic transmission caused by spastin loss of function are all restored to wild type when pak3 is simultaneously reduced. Neuronal overexpression of pak3 induces actin-rich thin projections, suggesting that it functions in vivo to promote filopodia during presynaptic terminal arborization. pak3 therefore regulates synapse development in vivo, and when mutated, suppresses the synaptic defects that result from spastin loss.


Assuntos
Adenosina Trifosfatases/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mutação/genética , Sinapses/metabolismo , Sinapses/ultraestrutura , Quinases Ativadas por p21/genética , Alelos , Animais , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/ultraestrutura , Epistasia Genética , Olho/metabolismo , Expressão Gênica , Estudo de Associação Genômica Ampla , Anotação de Sequência Molecular , Neurônios/metabolismo , Fenótipo , Pseudópodes/genética , Pseudópodes/metabolismo , Quinases Ativadas por p21/deficiência , Quinases Ativadas por p21/metabolismo
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