One third of human cancers are driven by oncogenic mutations in the KRAS loci. In particular, KRAS lesions are predominantly found in the most recalcitrant cancer types of internal organs such as pancreas, colon and lungs. A vast variety of signaling pathways is aberrantly activated downstream mutated KRAS, thus rendering tumours highly aggressive in terms of disease progression, chemoresistance and metastatic potential. Although several attempts have been made to design effective treatment strategies to eradicate these tumours, poor patients’ prognosis still remains a key issue in clinical oncology. One of the main limitations of therapies that selectively target kinase-signaling pathways is the emergence of secondary drug resistance. Therefore, new therapeutic approaches are based on combinatorial therapies, which concomitantly hit the tumour from different sides. Herein, we firstly show a synthetic lethal interaction of Smac-mimetic 83 (SM83) and Camptothecin (CPT) in presence of mutated KRAS employing a model of premalignant tumour cells. Mechanistically, we find that knock-in introduction of mutated KRAS upregulates Noxa via MAPK ERK2. At the same time, SM83, targeting the inhibitors of apoptosis proteins (IAPs) displaces caspase and enhances TNF-mediated cell death. Thus, specific KRAS mutations offer an unusual death-prone scenario in which, together with SM83, CPT efficiently triggers the mitochondrial apoptotic pathway in premalignant cells. Contrarily, in colorectal cancer settings the combination of SM83 and CPT is not lethal when KRAS is mutated suggesting that other pathways are aberrantly activated and render cancer cells resistant to death. Secondly, we propose a novel therapeutic strategy based on selective inhibition of Cyclin Dependent kinase 9 (CDK9) and concomitant induction of apoptosis via death ligand TRAIL in KRAS-mutated non-small cell lung cancer (NSCLC) cell lines. We indeed demonstrate that SNS-032, the most specific and clinically used inhibitor of CDK9, strongly synergizes with TRAIL in killing a panel of human TRAIL-resistant NSCLC cell lines. Moreover, we provide evidence of the efficacy of this novel combination in vivo. In fact, SNS-032 and TRAIL co-treatment totally eradicates established lung tumours. However, as the mice engrafted are immunosuppressed, this model does not provide any information regarding the interaction of the treatment with the murine immune system. Therefore, to further validate the efficacy of our novel combination, we establish an autochthonous mouse model of NSCLC. Preliminary in vitro treatments with TRAIL and CDK9 inhibitor confirm the efficacy of the combination; nevertheless, the setting of the in vivo experiments is still ongoing. Despite several combinatorial approaches have been so far entered clinical trials, patients often relapse and metastasis is still the main cause of cancer-related deaths especially in presence of mutated KRAS, which provides cancer cells with high metastasising potential. Therefore, we finally focus on the mechanisms by which KRAS-mutated tumours metastasize. Indeed, we point out that endogenous TRAIL and TRAIL-R2-mediated signaling is required to promote migration, invasion and metastasis via activation of Rac-1/PI3K signaling axis in KRAS-mutated cancers. Interestingly, we provide genetic in vivo evidence that mouse TRAIL-R is a key driver of cancer progression and metastasis. Significance: KRAS-mutated cancers are still refractory to current targeted therapies. Therefore, we propose two drug-combination approaches, which aim to induce cell death in tumour cells: whereas SM83 in combination with CPT acts at premalignant stages of cancer development, CDK9 inhibition is capable to overcome TRAIL resistance in NSCLC even in vivo, thereby supporting further preclinical investigations. Moreover, we provide new insights on the mechanisms regulating the metastatic process mediated by endogenous TRAIL/TRAIL-R2 pathway. We thus envisage both TRAIL and TRAIL-R2 as potential candidate-targets in clinic.

SMAC-MIMETIC- AND TRAIL- BASED COMBINATIONS AS THERAPEUTIC STRATEGIES FOR KRAS-MUTATED CANCERS / A. Conti ; relatore: C. Carlo-Stella ; correlatore: D. Delia ; coordinatore: M. Locati. - : . Università degli Studi di Milano, 2015 Jan 12. ((27. ciclo, Anno Accademico 2014. [10.13130/conti-annalisa_phd2014].

SMAC-MIMETIC- AND TRAIL- BASED COMBINATIONS AS THERAPEUTIC STRATEGIES FOR KRAS-MUTATED CANCERS

A. Conti
2015-01-12

Abstract

One third of human cancers are driven by oncogenic mutations in the KRAS loci. In particular, KRAS lesions are predominantly found in the most recalcitrant cancer types of internal organs such as pancreas, colon and lungs. A vast variety of signaling pathways is aberrantly activated downstream mutated KRAS, thus rendering tumours highly aggressive in terms of disease progression, chemoresistance and metastatic potential. Although several attempts have been made to design effective treatment strategies to eradicate these tumours, poor patients’ prognosis still remains a key issue in clinical oncology. One of the main limitations of therapies that selectively target kinase-signaling pathways is the emergence of secondary drug resistance. Therefore, new therapeutic approaches are based on combinatorial therapies, which concomitantly hit the tumour from different sides. Herein, we firstly show a synthetic lethal interaction of Smac-mimetic 83 (SM83) and Camptothecin (CPT) in presence of mutated KRAS employing a model of premalignant tumour cells. Mechanistically, we find that knock-in introduction of mutated KRAS upregulates Noxa via MAPK ERK2. At the same time, SM83, targeting the inhibitors of apoptosis proteins (IAPs) displaces caspase and enhances TNF-mediated cell death. Thus, specific KRAS mutations offer an unusual death-prone scenario in which, together with SM83, CPT efficiently triggers the mitochondrial apoptotic pathway in premalignant cells. Contrarily, in colorectal cancer settings the combination of SM83 and CPT is not lethal when KRAS is mutated suggesting that other pathways are aberrantly activated and render cancer cells resistant to death. Secondly, we propose a novel therapeutic strategy based on selective inhibition of Cyclin Dependent kinase 9 (CDK9) and concomitant induction of apoptosis via death ligand TRAIL in KRAS-mutated non-small cell lung cancer (NSCLC) cell lines. We indeed demonstrate that SNS-032, the most specific and clinically used inhibitor of CDK9, strongly synergizes with TRAIL in killing a panel of human TRAIL-resistant NSCLC cell lines. Moreover, we provide evidence of the efficacy of this novel combination in vivo. In fact, SNS-032 and TRAIL co-treatment totally eradicates established lung tumours. However, as the mice engrafted are immunosuppressed, this model does not provide any information regarding the interaction of the treatment with the murine immune system. Therefore, to further validate the efficacy of our novel combination, we establish an autochthonous mouse model of NSCLC. Preliminary in vitro treatments with TRAIL and CDK9 inhibitor confirm the efficacy of the combination; nevertheless, the setting of the in vivo experiments is still ongoing. Despite several combinatorial approaches have been so far entered clinical trials, patients often relapse and metastasis is still the main cause of cancer-related deaths especially in presence of mutated KRAS, which provides cancer cells with high metastasising potential. Therefore, we finally focus on the mechanisms by which KRAS-mutated tumours metastasize. Indeed, we point out that endogenous TRAIL and TRAIL-R2-mediated signaling is required to promote migration, invasion and metastasis via activation of Rac-1/PI3K signaling axis in KRAS-mutated cancers. Interestingly, we provide genetic in vivo evidence that mouse TRAIL-R is a key driver of cancer progression and metastasis. Significance: KRAS-mutated cancers are still refractory to current targeted therapies. Therefore, we propose two drug-combination approaches, which aim to induce cell death in tumour cells: whereas SM83 in combination with CPT acts at premalignant stages of cancer development, CDK9 inhibition is capable to overcome TRAIL resistance in NSCLC even in vivo, thereby supporting further preclinical investigations. Moreover, we provide new insights on the mechanisms regulating the metastatic process mediated by endogenous TRAIL/TRAIL-R2 pathway. We thus envisage both TRAIL and TRAIL-R2 as potential candidate-targets in clinic.
CARLO-STELLA, CARMELO
LOCATI, MASSIMO
Cancer ; mutated KRAS ; Smac-Mimetic ; TRAIL; drug-combinations
Settore BIO/13 - Biologia Applicata
Centro Interdipartimentale Studi Biomolecolari e Applicazioni Industriali -CISI
SMAC-MIMETIC- AND TRAIL- BASED COMBINATIONS AS THERAPEUTIC STRATEGIES FOR KRAS-MUTATED CANCERS / A. Conti ; relatore: C. Carlo-Stella ; correlatore: D. Delia ; coordinatore: M. Locati. - : . Università degli Studi di Milano, 2015 Jan 12. ((27. ciclo, Anno Accademico 2014. [10.13130/conti-annalisa_phd2014].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/249662
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