KEVETRIN: PRECLINICAL STUDY OF A NEW COMPOUND IN ACUTE MYELOID LEUKEMIA
Author(s):
Roberta Napolitano
,
Roberta Napolitano
Affiliations:
Bioscience Laboratory,IRCCS, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST),Meldola,Italy
Serena De Matteis
,
Serena De Matteis
Affiliations:
Bioscience Laboratory,IRCCS, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST),Meldola,Italy
Silvia Carloni
,
Silvia Carloni
Affiliations:
Bioscience Laboratory,IRCCS, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST),Meldola,Italy
Giorgia Simonetti
,
Giorgia Simonetti
Affiliations:
Department of Hematology and Oncological Sciences,L. and A. Seragnoli,Bologna,Italy
Gerardo Musuraca
,
Gerardo Musuraca
Affiliations:
Hematology Unit,IRCCS, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST),Meldola,Italy
Alessandro Lucchesi
,
Alessandro Lucchesi
Affiliations:
Hematology Unit,IRCCS, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST),Meldola,Italy
Daniele Calistri
,
Daniele Calistri
Affiliations:
Bioscience Laboratory,IRCCS, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST),Meldola,Italy
Antonio Cuneo
,
Antonio Cuneo
Affiliations:
Department of Medical Sciences,University of Ferrara-Arcispedale Sant'Anna,Ferrara,Italy
Krishna Menon
,
Krishna Menon
Affiliations:
Cellceutix Corporation,Beverly,United States
Giovanni Martinelli
Giovanni Martinelli
Affiliations:
Department of Hematology and Oncological Sciences,L. and A. Seragnoli,Bologna,Italy
(Abstract release date: 05/18/17) EHA Library. De Matteis S. 05/18/17; 180680; E904
Dr. Serena De Matteis
Dr. Serena De Matteis
Contributions
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Abstract

Abstract: E904

Type: Eposter Presentation

Background

Acute Myeloid Leukemia (AML) is a heterogeneous disorder defined by clonal expansion of immature myeloid cells that infiltrate bone marrow and other tissues. AML therapeutic strategies remain unchanged since 1970 and the majority of patients often eventually relapse and die due to disease progression. Tumor protein p53 transcription factor is a key regulator of several cellular pathways, such as DNA repair, cell cycle, apoptosis and angiogenesis. It is mutated in 8-14% of AML cases and its mutations are commonly associated with a complex karyotype. Kevetrin is a new molecule compound, proposed by Cellceutix, with the ability to target both wild type and mutant p53 tumors.

Aims
The aim of this project is to explore cellular and molecular alterations induced by Kevetrin, focusing on its role in the p53 pathway.

Methods

Kevetrin was kindly provided by Cellceutix, dissolved and stored at 4°C in sterile water in a 600 μg/ml stock solution, and diluted in medium immediately before use [concentration range in use 15-60 μg/ml]. Cell lines, MOLM-13 and KASUMI-1, were cultured in RPMI 1640 supplemented with 20% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. After 24 and 48 h of treatment MTS, Annexin-V, TUNEL, JC-1 and Active Caspase-3 assays were performed according to manufacturer’s instructions. Proteins were separated by polyacrylamide gel electrophoresis and transferred to 0.2 μm polyvinylidene fluoride membranes. Quantitative analysis was performed with Quantity One software. Statistical analysis was carried out using the paired and unpaired two-tailed Student’s t tests. p values < 0.05 were considered as significant.

Results
Our data indicate that Kevetrin exposure induces cell growth arrest, a great drop of mitochondrial membrane potential and a remarkable increment of Caspase-3 cleaved form, features that contribute to apoptotic cell death in the two cell lines. Cellular changes can be associated with a dose and time-dependent effect in the TP53 mutated cell line (KASUMI-1) but not in the wild type one (MOLM-13), in which we can observe an activity only after 48 h at the higher concentration. Regarding molecular alterations in KASUMI-1 we found a great p53 down-regulation, probably due to Hsp90 reduction, resulting in a less marked formation of the Hsp90-p53 oncogenic complex. We also found a down-regulated p53 active form (Ser15), a reduced expression of p53 targets, p21 and PUMA, and a down-regulation of SIRT-3, that cannot exert its inhibitory activity on p53. The MOLM-13 cell line showed a great p53 reduction, probably related to SIRT-3 up-regulation and Hsp90 down-regulation. Regarding p53 active form, we noticed slight variations in protein expression, suggesting a physiological response of the protein to cellular damage. In accordance with p53 activity, we observed a great up-regulation of p21, probably associated with a drug resistance mechanism; in contrast, PUMA protein was highly down-regulated, suggesting a p53-independent mechanism of action or a feedback regulation of the apoptotic process, after Caspase-3 activation (Figure). In order to better understand drug’s mechanism of action we are performing gene expression profiling after 48 h of treatment with Kevetrin 60 μg/ml.

Conclusion

Our results suggest Kevetrin is a promising new drug in AML patients treatment, both in wild type and, even more, in TP53 mutated tumors, through different molecular mechanisms, giving more therapeutic alternatives in the treatment of this disease.

Session topic: 3. Acute myeloid leukemia - Biology

Abstract: E904

Type: Eposter Presentation

Background

Acute Myeloid Leukemia (AML) is a heterogeneous disorder defined by clonal expansion of immature myeloid cells that infiltrate bone marrow and other tissues. AML therapeutic strategies remain unchanged since 1970 and the majority of patients often eventually relapse and die due to disease progression. Tumor protein p53 transcription factor is a key regulator of several cellular pathways, such as DNA repair, cell cycle, apoptosis and angiogenesis. It is mutated in 8-14% of AML cases and its mutations are commonly associated with a complex karyotype. Kevetrin is a new molecule compound, proposed by Cellceutix, with the ability to target both wild type and mutant p53 tumors.

Aims
The aim of this project is to explore cellular and molecular alterations induced by Kevetrin, focusing on its role in the p53 pathway.

Methods

Kevetrin was kindly provided by Cellceutix, dissolved and stored at 4°C in sterile water in a 600 μg/ml stock solution, and diluted in medium immediately before use [concentration range in use 15-60 μg/ml]. Cell lines, MOLM-13 and KASUMI-1, were cultured in RPMI 1640 supplemented with 20% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. After 24 and 48 h of treatment MTS, Annexin-V, TUNEL, JC-1 and Active Caspase-3 assays were performed according to manufacturer’s instructions. Proteins were separated by polyacrylamide gel electrophoresis and transferred to 0.2 μm polyvinylidene fluoride membranes. Quantitative analysis was performed with Quantity One software. Statistical analysis was carried out using the paired and unpaired two-tailed Student’s t tests. p values < 0.05 were considered as significant.

Results
Our data indicate that Kevetrin exposure induces cell growth arrest, a great drop of mitochondrial membrane potential and a remarkable increment of Caspase-3 cleaved form, features that contribute to apoptotic cell death in the two cell lines. Cellular changes can be associated with a dose and time-dependent effect in the TP53 mutated cell line (KASUMI-1) but not in the wild type one (MOLM-13), in which we can observe an activity only after 48 h at the higher concentration. Regarding molecular alterations in KASUMI-1 we found a great p53 down-regulation, probably due to Hsp90 reduction, resulting in a less marked formation of the Hsp90-p53 oncogenic complex. We also found a down-regulated p53 active form (Ser15), a reduced expression of p53 targets, p21 and PUMA, and a down-regulation of SIRT-3, that cannot exert its inhibitory activity on p53. The MOLM-13 cell line showed a great p53 reduction, probably related to SIRT-3 up-regulation and Hsp90 down-regulation. Regarding p53 active form, we noticed slight variations in protein expression, suggesting a physiological response of the protein to cellular damage. In accordance with p53 activity, we observed a great up-regulation of p21, probably associated with a drug resistance mechanism; in contrast, PUMA protein was highly down-regulated, suggesting a p53-independent mechanism of action or a feedback regulation of the apoptotic process, after Caspase-3 activation (Figure). In order to better understand drug’s mechanism of action we are performing gene expression profiling after 48 h of treatment with Kevetrin 60 μg/ml.

Conclusion

Our results suggest Kevetrin is a promising new drug in AML patients treatment, both in wild type and, even more, in TP53 mutated tumors, through different molecular mechanisms, giving more therapeutic alternatives in the treatment of this disease.

Session topic: 3. Acute myeloid leukemia - Biology

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