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JQ1 AND CURCUMIN COMBINED TREATMENT SHOWS SYNERGIC EFFECTS IN MLL-REARRANGED LEUKEMIA CELL LINES
Author(s):
Patrizia Vitullo
,
Patrizia Vitullo
Affiliations:
Oncohaematology,Bambino Gesù Children Hospital,Roma,Italy
Pier Paolo Leoncini
,
Pier Paolo Leoncini
Affiliations:
Oncohaematology,Bambino Gesù Children Hospital,Roma,Italy
Valeria Tocco
,
Valeria Tocco
Affiliations:
Oncohaematology,Bambino Gesù Children Hospital,Roma,Italy
Martina Pigazzi
,
Martina Pigazzi
Affiliations:
Pediatrics-Oncoematology,Univeristy of Padova,Padova,Italy
Katia D'Ovidio
,
Katia D'Ovidio
Affiliations:
Oncohaematology,Bambino Gesù Children Hospital,Roma,Italy
Daniela Montagna
,
Daniela Montagna
Affiliations:
Pediatrics,Policlinico San Matteo,Pavia,Italy
Rossella Rota
,
Rossella Rota
Affiliations:
Oncohaematology,Bambino Gesù Children Hospital,Roma,Italy
Alice Bertaina
Alice Bertaina
Affiliations:
Oncohaematology,Bambino Gesù Children Hospital,Roma,Italy
(Abstract release date: 05/18/17) EHA Library. Vitullo P. 05/18/17; 182379; PB1665
Patrizia Vitullo
Patrizia Vitullo
Contributions
PDF Abstract
Abstract

Abstract: PB1665

Type: Publication Only

Background

MLL-rearranged leukemia accounts for ∼70% of infant and ∼10% adult acute leukemias, featuring a particularly poor prognosis and high risk of relapse. Our main field of study is AML, in which nearly 50% of total cases accounts for t(9;11) translocation, the remaining 50% predominantly includes t(6;11)(q27;q23), t(10;11)(p12;q23), t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3). A 2% of AML total cases, however, is characterized by t(4;11) translocation, which is a marker of bad prognosis and it’s, so far, poorly characterized. A key feature of MLL-rearranged leukemia is cMyc overexpression, a well-known oncogene involved in several types of cancer. JQ1 is a novel molecule, which prevents cMYC expression binding an important bromodomain protein, BRD4. Moreover, Curcumin, a natural compound, inhibits HATs enzymes preventing lysine 14 acetylation on histone H3 (AcH3K14), a particular residue which is bind by BRD4 to exert its function.

Aims
We would like to explore a potential synergic effect between JQ1 and Curcumin molecules in the attempt to develop a novel therapeutic alternative to standard chemotherapy and to deeply investigate features underlying the molecular pathogenesis in pediatric MLL-rearranged pediatric AML.

Methods

Four human leukemia cell lines with MLL fusion protein have been employed in this study: RS4:11, MV4:11 expressing MLL-AF4 and THP1, MOLM13 expressing MLL-AF9 fusion genes. 5μM and 10μM Curcumin were used to treat MLL-AF4 and MLL-AF9 cell lines respectively, while 250nM JQ1 was used to treat all the cells lines. After 2 days of treatment, either with single and combined drugs, cell number quantification, based on metabolic activity, was detected through XTT assay. In order to assess the cMYC, CDKN1A, BCL2 transcripts levels and mir-99a expression a quantitative RT-PCR analysis was carried out, while we used western blotting to detect the expression of cMYC, PARP, Caspase3 and AcH3K14. Apoptosis and cell cycle were evaluated by flow cytometric analysis.

Results
In apoptosis analysis, a synergic effect was detected for all 4 cell lines, similarly cell cycle evaluation showed a significant accumulation of cells at SubG1 phase (2-8 fold) (Figure). XTT metabolic assay showed a reduction in proliferation percentage: 65±5 for curcumin and JQ1 single treatment and 59±5 for combination of drugs in both MLL-AF4 cell lines, meanwhile in MOLM13 cells it was 64±2 and 87±2 for curcumin and JQ1, respectively and 76±2 for their combination (P<0.005). The THP1 cells did not shown any significant modulation in the proliferation. We decided to focus our study on t(4:11) translocated cells, considering the more intense effect of the combined drugs on previous analysis. qRT-PCR and western blot experiments revealed a synergic effect of the 2 experimental drugs on both apoptosis and proliferation gene related (bcl2, caspase3, Parp, cdkn1a) as well as on the direct targets of the drugs (cMyc, AcH3K14). Finally, in MLL-AF4 cell lines, curcumin and JQ1 together induced a significant decrease in mir-99a expression.

Conclusion
Our data demonstrated that curcumin and JQ1, inhibiting HATs and BRD4 respectively, exert a more intense synergic effect on MLL-AF4 than in MLL-AF9 cells. Increased apoptosis together with a reduced proliferation rate, prompted us to investigate on molecular pathway in which targets of these drugs are involved. Intriguingly, we found a significant decrease in cMyc, bcl2 and AcH3K14 expression, confirming that both curcumin and JQ1 have a synergic effect. Additionally, we revealed a significant reduced expression of mir-99a, a well know oncomiR reported to act as negative regulator of differentiation and involved in drug-resistance, typically up-regulated in pediatric AML and ALL.

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): Pediatric, MYC, Leukemia cell line, 11q23

Abstract: PB1665

Type: Publication Only

Background

MLL-rearranged leukemia accounts for ∼70% of infant and ∼10% adult acute leukemias, featuring a particularly poor prognosis and high risk of relapse. Our main field of study is AML, in which nearly 50% of total cases accounts for t(9;11) translocation, the remaining 50% predominantly includes t(6;11)(q27;q23), t(10;11)(p12;q23), t(11;19)(q23;p13.1) and t(11;19)(q23;p13.3). A 2% of AML total cases, however, is characterized by t(4;11) translocation, which is a marker of bad prognosis and it’s, so far, poorly characterized. A key feature of MLL-rearranged leukemia is cMyc overexpression, a well-known oncogene involved in several types of cancer. JQ1 is a novel molecule, which prevents cMYC expression binding an important bromodomain protein, BRD4. Moreover, Curcumin, a natural compound, inhibits HATs enzymes preventing lysine 14 acetylation on histone H3 (AcH3K14), a particular residue which is bind by BRD4 to exert its function.

Aims
We would like to explore a potential synergic effect between JQ1 and Curcumin molecules in the attempt to develop a novel therapeutic alternative to standard chemotherapy and to deeply investigate features underlying the molecular pathogenesis in pediatric MLL-rearranged pediatric AML.

Methods

Four human leukemia cell lines with MLL fusion protein have been employed in this study: RS4:11, MV4:11 expressing MLL-AF4 and THP1, MOLM13 expressing MLL-AF9 fusion genes. 5μM and 10μM Curcumin were used to treat MLL-AF4 and MLL-AF9 cell lines respectively, while 250nM JQ1 was used to treat all the cells lines. After 2 days of treatment, either with single and combined drugs, cell number quantification, based on metabolic activity, was detected through XTT assay. In order to assess the cMYC, CDKN1A, BCL2 transcripts levels and mir-99a expression a quantitative RT-PCR analysis was carried out, while we used western blotting to detect the expression of cMYC, PARP, Caspase3 and AcH3K14. Apoptosis and cell cycle were evaluated by flow cytometric analysis.

Results
In apoptosis analysis, a synergic effect was detected for all 4 cell lines, similarly cell cycle evaluation showed a significant accumulation of cells at SubG1 phase (2-8 fold) (Figure). XTT metabolic assay showed a reduction in proliferation percentage: 65±5 for curcumin and JQ1 single treatment and 59±5 for combination of drugs in both MLL-AF4 cell lines, meanwhile in MOLM13 cells it was 64±2 and 87±2 for curcumin and JQ1, respectively and 76±2 for their combination (P<0.005). The THP1 cells did not shown any significant modulation in the proliferation. We decided to focus our study on t(4:11) translocated cells, considering the more intense effect of the combined drugs on previous analysis. qRT-PCR and western blot experiments revealed a synergic effect of the 2 experimental drugs on both apoptosis and proliferation gene related (bcl2, caspase3, Parp, cdkn1a) as well as on the direct targets of the drugs (cMyc, AcH3K14). Finally, in MLL-AF4 cell lines, curcumin and JQ1 together induced a significant decrease in mir-99a expression.

Conclusion
Our data demonstrated that curcumin and JQ1, inhibiting HATs and BRD4 respectively, exert a more intense synergic effect on MLL-AF4 than in MLL-AF9 cells. Increased apoptosis together with a reduced proliferation rate, prompted us to investigate on molecular pathway in which targets of these drugs are involved. Intriguingly, we found a significant decrease in cMyc, bcl2 and AcH3K14 expression, confirming that both curcumin and JQ1 have a synergic effect. Additionally, we revealed a significant reduced expression of mir-99a, a well know oncomiR reported to act as negative regulator of differentiation and involved in drug-resistance, typically up-regulated in pediatric AML and ALL.

Session topic: 3. Acute myeloid leukemia - Biology

Keyword(s): Pediatric, MYC, Leukemia cell line, 11q23

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