RESUMO
Glioblastoma is the most common primary brain tumor in adults, characterized by an inherent aggressivity and resistance to treatment leading to poor prognoses. While some resistance mechanisms have been elucidated, a deeper understanding of these mechanisms is needed to increase therapeutic efficacy. In this study we first discovered glial-cell derived neurotrophic factor (GDNF) to be upregulated in patient-derived glioblastoma spheroid cultures after chemotherapeutic temozolomide treatment, through RNA-Seq experiments. Therefore, we investigated the role of the GDNF/GDNF receptor alpha 1 (GFRA1) signaling pathway as a resistance mechanism to chemotherapy with temozolomide and lomustine, as well as irradiation using patient-derived glioblastoma spheroid cultures. With qPCR experiments we showed a consistent upregulation of GDNF and its primary receptor GFRA1 following all three lines of treatment. Moreover, CRISPR/Cas9 knock-outs of GDNF in two patient-derived models sensitized these cells to chemotherapy treatment, but not radiotherapy. The increased sensitivity was completely reversed by the addition of exogeneous GDNF, confirming the key role of this factor in chemoresistance. Finally, a CRISPR KO of GFRA1 demonstrated a similar increased sensitivity to temozolomide and lomustine treatment, as well as radiotherapy. Together, our findings support the role of the GDNF/GFRA1 signaling pathway in glioblastoma chemo and radioresistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Glioblastoma , Tolerância a Radiação , Transdução de Sinais , Temozolomida , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/radioterapia , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Resistencia a Medicamentos Antineoplásicos/genética , Temozolomida/farmacologia , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos dos fármacos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lomustina/farmacologia , Esferoides Celulares/metabolismo , Esferoides Celulares/efeitos dos fármacosRESUMO
How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.
Assuntos
Carcinoma Ductal Pancreático , MAP Quinases Reguladas por Sinal Extracelular , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Mutação , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Transcriptoma , Animais , Humanos , Camundongos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células HEK293RESUMO
Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for improved therapeutic combinations and their predictive biomarkers. Starting from a CRISPR knockout screen, we identified that loss of RB1 in TNBC or HGSC cells generates a synthetic lethal dependency on casein kinase 2 (CK2) for surviving the treatment with replication-perturbing therapeutics such as carboplatin, gemcitabine, or PARP inhibitors. CK2 inhibition in RB1-deficient cells resulted in the degradation of another RB family cell cycle regulator, p130, which led to S phase accumulation, micronuclei formation, and accelerated PARP inhibition-induced aneuploidy and mitotic cell death. CK2 inhibition was also effective in primary patient-derived cells. It selectively prevented the regrowth of RB1-deficient patient HGSC organoids after treatment with carboplatin or niraparib. As about 25% of HGSCs and 40% of TNBCs have lost RB1 expression, CK2 inhibition is a promising approach to overcome resistance to standard therapeutics in large strata of patients.
Assuntos
Caseína Quinase II , Proteínas de Ligação a Retinoblastoma , Humanos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/metabolismo , Caseína Quinase II/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Feminino , Linhagem Celular Tumoral , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Carboplatina/farmacologia , Mutações Sintéticas Letais , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antineoplásicos/farmacologiaRESUMO
BCR::ABL1-independent pathways contribute to primary resistance to tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) and play a role in leukemic stem cell persistence. Here, we perform ex vivo drug screening of CML CD34+ leukemic stem/progenitor cells using 100 single drugs and TKI-drug combinations and identify sensitivities to Wee1, MDM2, and BCL2 inhibitors. These agents effectively inhibit primitive CD34+CD38- CML cells and demonstrate potent synergies when combined with TKIs. Flow-cytometry-based drug screening identifies mepacrine to induce differentiation of CD34+CD38- cells. We employ genome-wide CRISPR-Cas9 screening for six drugs, and mediator complex, apoptosis, and erythroid-lineage-related genes are identified as key resistance hits for TKIs, whereas the Wee1 inhibitor AZD1775 and mepacrine exhibit distinct resistance profiles. KCTD5, a consistent TKI-resistance-conferring gene, is found to mediate TKI-induced BCR::ABL1 ubiquitination. In summary, we delineate potential mechanisms for primary TKI resistance and non-BCR::ABL1-targeting drugs, offering insights for optimizing CML treatment.