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1.
J Biol Chem ; 298(11): 102550, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-36183837

RESUMEN

BRCA1/2-deficient ovarian carcinoma (OC) has been shown to be particularly sensitive to poly (ADP-ribose) polymerase inhibitors (PARPis). Furthermore, BRCA1/2 mutation status is currently used as a predictive biomarker for PARPi therapy. Despite providing a major clinical benefit to the majority of patients, a significant proportion of BRCA1/2-deficient OC tumors do not respond to PARPis for reasons that are incompletely understood. Using an integrated chemical, phospho- and ADP-ribosylation proteomics approach, we sought here to develop additional mechanism-based biomarker candidates for PARPi therapy in OC and identify new targets for combination therapy to overcome primary resistance. Using chemical proteomics with PARPi baits in a BRCA1-isogenic OC cell line pair, as well as patient-derived BRCA1-proficient and BRCA1-deficient tumor samples, and subsequent validation by coimmunoprecipitation, we showed differential PARP1 and PARP2 protein complex composition in PARPi-sensitive, BRCA1-deficient UWB1.289 (UWB) cells compared to PARPi-insensitive, BRCA1-reconstituted UWB1.289+BRCA1 (UWB+B) cells. In addition, global phosphoproteomics and ADP-ribosylation proteomics furthermore revealed that the PARPi rucaparib induced the cell cycle pathway and nonhomologous end joining (NHEJ) pathway in UWB cells but downregulated ErbB signaling in UWB+B cells. In addition, we observed AKT PARylation and prosurvival AKT-mTOR signaling in UWB+B cells after PARPi treatment. Consistently, we found the synergy of PARPis with DNAPK or AKT inhibitors was more pronounced in UWB+B cells, highlighting these pathways as actionable vulnerabilities. In conclusion, we demonstrate the combination of chemical proteomics, phosphoproteomics, and ADP-ribosylation proteomics can identify differential PARP1/2 complexes and diverse, but actionable, drug compensatory signaling in OC.


Asunto(s)
Neoplasias Ováricas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Femenino , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Proteómica , Proteínas Proto-Oncogénicas c-akt , Resistencia a Antineoplásicos , Línea Celular Tumoral , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología
2.
Chembiochem ; 24(11): e202200766, 2023 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-36922348

RESUMEN

Metastasis poses a major challenge in cancer management, including EML4-ALK-rearranged non-small cell lung cancer (NSCLC). As cell migration is a critical step during metastasis, we assessed the anti-migratory activities of several clinical ALK inhibitors in NSCLC cells and observed differential anti-migratory capabilities despite similar ALK inhibition, with brigatinib displaying superior anti-migratory effects over other ALK inhibitors. Applying an unbiased in situ mass spectrometry-based chemoproteomics approach, we determined the proteome-wide target profile of brigatinib in EML4-ALK+ NSCLC cells. Dose-dependent and cross-competitive chemoproteomics suggested MARK2 and MARK3 as relevant brigatinib kinase targets. Functional validation showed that combined pharmacological inhibition or genetic modulation of MARK2/3 inhibited cell migration. Consistently, brigatinib treatment induced inhibitory YAP1 phosphorylation downstream of MARK2/3. Collectively, our data suggest that brigatinib exhibits unusual cross-phenotype polypharmacology as, despite similar efficacy for inhibiting EML4-ALK-dependent cell proliferation as other ALK inhibitors, it more effectively prevented migration of NSCLC cells due to co-targeting of MARK2/3.


Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Quinasa de Linfoma Anaplásico/uso terapéutico , Compuestos Organofosforados/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Movimiento Celular , Proteínas Serina-Treonina Quinasas
3.
Am J Transplant ; 22(3): 717-730, 2022 03.
Artículo en Inglés | MEDLINE | ID: mdl-34668635

RESUMEN

Prevention of allograft rejection often requires lifelong immune suppression, risking broad impairment of host immunity. Nonselective inhibition of host T cell function increases recipient risk of opportunistic infections and secondary malignancies. Here we demonstrate that AJI-100, a dual inhibitor of JAK2 and Aurora kinase A, ameliorates skin graft rejection by human T cells and provides durable allo-inactivation. AJI-100 significantly reduces the frequency of skin-homing CLA+ donor T cells, limiting allograft invasion and tissue destruction by T effectors. AJI-100 also suppresses pathogenic Th1 and Th17 cells in the spleen yet spares beneficial regulatory T cells. We show dual JAK2/Aurora kinase A blockade enhances human type 2 innate lymphoid cell (ILC2) responses, which are capable of tissue repair. ILC2 differentiation mediated by GATA3 requires STAT5 phosphorylation (pSTAT5) but is opposed by STAT3. Further, we demonstrate that Aurora kinase A activation correlates with low pSTAT5 in ILC2s. Importantly, AJI-100 maintains pSTAT5 levels in ILC2s by blocking Aurora kinase A and reduces interference by STAT3. Therefore, combined JAK2/Aurora kinase A inhibition is an innovative strategy to merge immune suppression with tissue repair after transplantation.


Asunto(s)
Aurora Quinasa A , Inmunidad Innata , Animales , Aurora Quinasa A/metabolismo , Rechazo de Injerto/etiología , Rechazo de Injerto/prevención & control , Humanos , Janus Quinasa 2 , Ratones , Ratones Endogámicos C57BL , Células Th17 , Trasplante Homólogo
4.
Blood ; 136(7): 857-870, 2020 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-32403132

RESUMEN

Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known "neosubstrates," such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Linfocitos T CD8-positivos/fisiología , Metabolismo Energético/genética , Activación de Linfocitos/genética , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Inmunomodulación/genética , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos
5.
J Med Chem ; 64(4): 2228-2241, 2021 02 25.
Artículo en Inglés | MEDLINE | ID: mdl-33570945

RESUMEN

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.


Asunto(s)
Janus Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirazoles/farmacología , Pirrolidinas/farmacología , Sulfonamidas/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Janus Quinasa 2/metabolismo , Estructura Molecular , Nitrilos , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Pirazoles/química , Pirazoles/metabolismo , Pirimidinas , Pirrolidinas/química , Pirrolidinas/metabolismo , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/metabolismo
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