RECOVERY OF TUMOUR SUPPRESSOR FUNCTION OF NOTCH3 BY AN ASPERGILLUM DERIVED MYCOTOXIN IN CHRONIC LYMPHOCYTIC LEUKAEMIA (CLL)
(Abstract release date: 05/19/16)
EHA Library. Hubmann R. 06/09/16; 135362; LB2251
Dr. Rainer Hubmann
Contributions
Contributions
Abstract
Abstract: LB2251
Type: Eposter Presentation
Background
Deregulation of NOTCH2 signalling is critically involved in CLL leukemogenesis. We have recently shown that CLL cells are less sensitive to γ-secretase inhibitors (GSI) indicative for the expression of constitutively active NOTCH2 forms which do not depend on γ-secretase for processing and function. In contrast, targeting CLL cells with the Aspergillum derived NOTCH2 transactivation inhibitor gliotoxin efficiently induced apoptosis in the leukemic cells by a mechanism which appeared to involve the induction of NOTCH3.
Aims
To gain deeper insight into the function of NOTCH3 in CLL cells, we analysed NOTCH3 expression in relation to spontaneous and gliotoxin induced apoptosis and tested the effect of NOTCH3 inhibition on CLL cell viability.
Methods
The NOTCH3 gene was inhibited by GSI (RO4929097) or by siRNA mediated gene silencing. Apoptosis was calculated as the sum of PI-/AnxV+ (early apoptosis) and PI+/AnxV+ (late apoptosis) cells. The expression of NOTCH and apoptosis related genes was monitored by RT-PCR and FACS analysis.
Results
Results showed that inhibition of NOTCH2 signaling by gliotoxin led to the concomitant induction of NOTCH3 signalling as indicated by the induced co-expression of NOTCH3 and its ligands JAG2 (in unstimulated CLL cells) or DLL1 (in PMA activated CLL cells) at the mRNA level. Gliotoxin induced a tumour suppressive NOTCH3 signalling signature as demonstrated by the induction of the apoptosis regulating NOTCH3 target genes HEY1, NR4A1, and CDKN1A (p21) in the pre-apoptotic phase. In contrast, the anti-apoptotic genes NOTCH2, FCER2 (CD23), and AKT1 were inhibited by gliotoxin.We then tested the effect of gliotoxin and GSI on induction of apoptosis. Gliotoxin induced apoptosis in CLL cells (mean % ± SD: 85 ± 10 % versus 13 ± 5 %), while GSI (RO4929097) mediated NOTCH3 inhibition was associated with a decrease in spontaneous (7 ± 3 % versus 13 ± 5 %) and gliotoxin induced apoptosis (51 ±7 % versus 85 ± 10 %) in the leukemic cells.To directly confirm that NOTCH3 has a pro-apoptotic role in CLL, we silenced NOTCH3 by RNA interference. After 3 days in culture, NOTCH3 siRNA decreased the apoptotic effect of gliotoxin leading to a 3.5-fold increase in the percentage of living CLL cells. Inhibition of NOTCH3 by siRNA was also associated with an increased surface expression of CD23, a downstream target of NOTCH2. These data show that the inhibition of NOTCH3 may lead to the upregulation of NOTCH2/CD23 axis and resistance to apoptosis. This reciprocal regulation of NOTCH2 and NOTCH3 together with their opposite effects on cell viability strongly suggests that these two NOTCH receptors have different roles in the regulation of the viability of CLL cells. Interestingly, NOTCH3 mRNA could be detected in patients with early stage of the disease (Rai 0-II) but not in Rai III-IV. Thus, in CLL cells, constitutive active NOTCH2 signalling might suppress pro-apoptotic NOTCH3 signalling enabling the expansion of the malignant clone particularly in advanced stages of the disease.
Conclusion
In summary, these novel data indicate that selective targeting oncogenic NOTCH2 might recover a pro-apoptotic NOTCH3 activity which should be considered in the design of therapies aimed to target the NOTCH pathway in CLL cells. Moreover, this data points for the first time to a tumour suppressive function for NOTCH3 in CLL.
Session topic: E-poster
Keyword(s): Apoptosis, Chronic lymphocytic leukemia, Notch signaling, Therapy
Type: Eposter Presentation
Background
Deregulation of NOTCH2 signalling is critically involved in CLL leukemogenesis. We have recently shown that CLL cells are less sensitive to γ-secretase inhibitors (GSI) indicative for the expression of constitutively active NOTCH2 forms which do not depend on γ-secretase for processing and function. In contrast, targeting CLL cells with the Aspergillum derived NOTCH2 transactivation inhibitor gliotoxin efficiently induced apoptosis in the leukemic cells by a mechanism which appeared to involve the induction of NOTCH3.
Aims
To gain deeper insight into the function of NOTCH3 in CLL cells, we analysed NOTCH3 expression in relation to spontaneous and gliotoxin induced apoptosis and tested the effect of NOTCH3 inhibition on CLL cell viability.
Methods
The NOTCH3 gene was inhibited by GSI (RO4929097) or by siRNA mediated gene silencing. Apoptosis was calculated as the sum of PI-/AnxV+ (early apoptosis) and PI+/AnxV+ (late apoptosis) cells. The expression of NOTCH and apoptosis related genes was monitored by RT-PCR and FACS analysis.
Results
Results showed that inhibition of NOTCH2 signaling by gliotoxin led to the concomitant induction of NOTCH3 signalling as indicated by the induced co-expression of NOTCH3 and its ligands JAG2 (in unstimulated CLL cells) or DLL1 (in PMA activated CLL cells) at the mRNA level. Gliotoxin induced a tumour suppressive NOTCH3 signalling signature as demonstrated by the induction of the apoptosis regulating NOTCH3 target genes HEY1, NR4A1, and CDKN1A (p21) in the pre-apoptotic phase. In contrast, the anti-apoptotic genes NOTCH2, FCER2 (CD23), and AKT1 were inhibited by gliotoxin.We then tested the effect of gliotoxin and GSI on induction of apoptosis. Gliotoxin induced apoptosis in CLL cells (mean % ± SD: 85 ± 10 % versus 13 ± 5 %), while GSI (RO4929097) mediated NOTCH3 inhibition was associated with a decrease in spontaneous (7 ± 3 % versus 13 ± 5 %) and gliotoxin induced apoptosis (51 ±7 % versus 85 ± 10 %) in the leukemic cells.To directly confirm that NOTCH3 has a pro-apoptotic role in CLL, we silenced NOTCH3 by RNA interference. After 3 days in culture, NOTCH3 siRNA decreased the apoptotic effect of gliotoxin leading to a 3.5-fold increase in the percentage of living CLL cells. Inhibition of NOTCH3 by siRNA was also associated with an increased surface expression of CD23, a downstream target of NOTCH2. These data show that the inhibition of NOTCH3 may lead to the upregulation of NOTCH2/CD23 axis and resistance to apoptosis. This reciprocal regulation of NOTCH2 and NOTCH3 together with their opposite effects on cell viability strongly suggests that these two NOTCH receptors have different roles in the regulation of the viability of CLL cells. Interestingly, NOTCH3 mRNA could be detected in patients with early stage of the disease (Rai 0-II) but not in Rai III-IV. Thus, in CLL cells, constitutive active NOTCH2 signalling might suppress pro-apoptotic NOTCH3 signalling enabling the expansion of the malignant clone particularly in advanced stages of the disease.
Conclusion
In summary, these novel data indicate that selective targeting oncogenic NOTCH2 might recover a pro-apoptotic NOTCH3 activity which should be considered in the design of therapies aimed to target the NOTCH pathway in CLL cells. Moreover, this data points for the first time to a tumour suppressive function for NOTCH3 in CLL.
Session topic: E-poster
Keyword(s): Apoptosis, Chronic lymphocytic leukemia, Notch signaling, Therapy
Abstract: LB2251
Type: Eposter Presentation
Background
Deregulation of NOTCH2 signalling is critically involved in CLL leukemogenesis. We have recently shown that CLL cells are less sensitive to γ-secretase inhibitors (GSI) indicative for the expression of constitutively active NOTCH2 forms which do not depend on γ-secretase for processing and function. In contrast, targeting CLL cells with the Aspergillum derived NOTCH2 transactivation inhibitor gliotoxin efficiently induced apoptosis in the leukemic cells by a mechanism which appeared to involve the induction of NOTCH3.
Aims
To gain deeper insight into the function of NOTCH3 in CLL cells, we analysed NOTCH3 expression in relation to spontaneous and gliotoxin induced apoptosis and tested the effect of NOTCH3 inhibition on CLL cell viability.
Methods
The NOTCH3 gene was inhibited by GSI (RO4929097) or by siRNA mediated gene silencing. Apoptosis was calculated as the sum of PI-/AnxV+ (early apoptosis) and PI+/AnxV+ (late apoptosis) cells. The expression of NOTCH and apoptosis related genes was monitored by RT-PCR and FACS analysis.
Results
Results showed that inhibition of NOTCH2 signaling by gliotoxin led to the concomitant induction of NOTCH3 signalling as indicated by the induced co-expression of NOTCH3 and its ligands JAG2 (in unstimulated CLL cells) or DLL1 (in PMA activated CLL cells) at the mRNA level. Gliotoxin induced a tumour suppressive NOTCH3 signalling signature as demonstrated by the induction of the apoptosis regulating NOTCH3 target genes HEY1, NR4A1, and CDKN1A (p21) in the pre-apoptotic phase. In contrast, the anti-apoptotic genes NOTCH2, FCER2 (CD23), and AKT1 were inhibited by gliotoxin.We then tested the effect of gliotoxin and GSI on induction of apoptosis. Gliotoxin induced apoptosis in CLL cells (mean % ± SD: 85 ± 10 % versus 13 ± 5 %), while GSI (RO4929097) mediated NOTCH3 inhibition was associated with a decrease in spontaneous (7 ± 3 % versus 13 ± 5 %) and gliotoxin induced apoptosis (51 ±7 % versus 85 ± 10 %) in the leukemic cells.To directly confirm that NOTCH3 has a pro-apoptotic role in CLL, we silenced NOTCH3 by RNA interference. After 3 days in culture, NOTCH3 siRNA decreased the apoptotic effect of gliotoxin leading to a 3.5-fold increase in the percentage of living CLL cells. Inhibition of NOTCH3 by siRNA was also associated with an increased surface expression of CD23, a downstream target of NOTCH2. These data show that the inhibition of NOTCH3 may lead to the upregulation of NOTCH2/CD23 axis and resistance to apoptosis. This reciprocal regulation of NOTCH2 and NOTCH3 together with their opposite effects on cell viability strongly suggests that these two NOTCH receptors have different roles in the regulation of the viability of CLL cells. Interestingly, NOTCH3 mRNA could be detected in patients with early stage of the disease (Rai 0-II) but not in Rai III-IV. Thus, in CLL cells, constitutive active NOTCH2 signalling might suppress pro-apoptotic NOTCH3 signalling enabling the expansion of the malignant clone particularly in advanced stages of the disease.
Conclusion
In summary, these novel data indicate that selective targeting oncogenic NOTCH2 might recover a pro-apoptotic NOTCH3 activity which should be considered in the design of therapies aimed to target the NOTCH pathway in CLL cells. Moreover, this data points for the first time to a tumour suppressive function for NOTCH3 in CLL.
Session topic: E-poster
Keyword(s): Apoptosis, Chronic lymphocytic leukemia, Notch signaling, Therapy
Type: Eposter Presentation
Background
Deregulation of NOTCH2 signalling is critically involved in CLL leukemogenesis. We have recently shown that CLL cells are less sensitive to γ-secretase inhibitors (GSI) indicative for the expression of constitutively active NOTCH2 forms which do not depend on γ-secretase for processing and function. In contrast, targeting CLL cells with the Aspergillum derived NOTCH2 transactivation inhibitor gliotoxin efficiently induced apoptosis in the leukemic cells by a mechanism which appeared to involve the induction of NOTCH3.
Aims
To gain deeper insight into the function of NOTCH3 in CLL cells, we analysed NOTCH3 expression in relation to spontaneous and gliotoxin induced apoptosis and tested the effect of NOTCH3 inhibition on CLL cell viability.
Methods
The NOTCH3 gene was inhibited by GSI (RO4929097) or by siRNA mediated gene silencing. Apoptosis was calculated as the sum of PI-/AnxV+ (early apoptosis) and PI+/AnxV+ (late apoptosis) cells. The expression of NOTCH and apoptosis related genes was monitored by RT-PCR and FACS analysis.
Results
Results showed that inhibition of NOTCH2 signaling by gliotoxin led to the concomitant induction of NOTCH3 signalling as indicated by the induced co-expression of NOTCH3 and its ligands JAG2 (in unstimulated CLL cells) or DLL1 (in PMA activated CLL cells) at the mRNA level. Gliotoxin induced a tumour suppressive NOTCH3 signalling signature as demonstrated by the induction of the apoptosis regulating NOTCH3 target genes HEY1, NR4A1, and CDKN1A (p21) in the pre-apoptotic phase. In contrast, the anti-apoptotic genes NOTCH2, FCER2 (CD23), and AKT1 were inhibited by gliotoxin.We then tested the effect of gliotoxin and GSI on induction of apoptosis. Gliotoxin induced apoptosis in CLL cells (mean % ± SD: 85 ± 10 % versus 13 ± 5 %), while GSI (RO4929097) mediated NOTCH3 inhibition was associated with a decrease in spontaneous (7 ± 3 % versus 13 ± 5 %) and gliotoxin induced apoptosis (51 ±7 % versus 85 ± 10 %) in the leukemic cells.To directly confirm that NOTCH3 has a pro-apoptotic role in CLL, we silenced NOTCH3 by RNA interference. After 3 days in culture, NOTCH3 siRNA decreased the apoptotic effect of gliotoxin leading to a 3.5-fold increase in the percentage of living CLL cells. Inhibition of NOTCH3 by siRNA was also associated with an increased surface expression of CD23, a downstream target of NOTCH2. These data show that the inhibition of NOTCH3 may lead to the upregulation of NOTCH2/CD23 axis and resistance to apoptosis. This reciprocal regulation of NOTCH2 and NOTCH3 together with their opposite effects on cell viability strongly suggests that these two NOTCH receptors have different roles in the regulation of the viability of CLL cells. Interestingly, NOTCH3 mRNA could be detected in patients with early stage of the disease (Rai 0-II) but not in Rai III-IV. Thus, in CLL cells, constitutive active NOTCH2 signalling might suppress pro-apoptotic NOTCH3 signalling enabling the expansion of the malignant clone particularly in advanced stages of the disease.
Conclusion
In summary, these novel data indicate that selective targeting oncogenic NOTCH2 might recover a pro-apoptotic NOTCH3 activity which should be considered in the design of therapies aimed to target the NOTCH pathway in CLL cells. Moreover, this data points for the first time to a tumour suppressive function for NOTCH3 in CLL.
Session topic: E-poster
Keyword(s): Apoptosis, Chronic lymphocytic leukemia, Notch signaling, Therapy
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