CHEMO-GENOMIC INTERROGATION OF PRIMARY ACUTE MYELOID LEUKEMIA WITH BIALLELIC CEBPA MUTATIONS REVEALS RECURRENT CSF3R MUTATIONS AND SUBGROUP SENSITIVITY TO JAK INHIBITORS.
(Abstract release date: 05/19/16)
EHA Library. Lavallée V. 06/12/16; 135298; S804
Dr. Vincent-Philippe Lavallée
Contributions
Contributions
Abstract
Abstract: S804
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:15 - 08:30
Location: Hall A3
Background
Acute myeloid leukemias (AML) with CEBPA mutations define a provisional entity in the WHO 2008 classification. Patients with biallelic CEBPA (CEBPAbi) mutations comprising N-terminal frameshift and C-terminal in frame mutations (hereafter termed typical CEBPAbi AML) characteristically present a normal karyotype and have a favorable clinical outcome. In contrast, characteristics of samples with other combinations of mutations (atypical CEBPAbi AML) are less well established.
Aims
Using the RNA-sequencing data from 415 primary AMLs in Leucegene cohort, we aimed to refine the transcriptomic and mutational landscape of 14 CEBPAbi AML samples (7 typical and 7 atypical) present in this collection, and to interrogate the novel mutations identified in this subgroup in a targeted chemical inhibitor screen.
Methods
We performed RNA-sequencing, comparative transcriptomic analysis, mutation detection, cell culture and chemical screening using the methods previously reported (Lavallée et al, Nature Genetics, 2015).
Results
The 7 typical CEBPAbi specimens are best characterized by 95 differentially expressed genes. Using this gene expression profile (GEP), we next performed a principal component analysis and determined that 4/7 atypical CEBPAbi samples clustered with typical CEBPAbi (hereafter termed GEP+), while 3/7 atypical CEBPAbi samples did not (GEP-). Identification of low HOXA9 expression alone was sufficient to discriminate between GEP+ and GEP- atypical CEBPA AML (> 300 fold median HOXA9 expression ratios).In our cohort of CEBPAbi AML we identified 23 mutated genes, including the previously reported mutations in WT1 and GATA2 in 3/14 (21% each). The most frequent mutations in CEBPAbi AML was the activating CSF3R T618I mutation present in 29% (4/14) of this subgroup compared to only 3/401 CSF3R mutated samples in other AML subtypes (p < 0.0001). CSF3R encodes the granulocyte colony stimulating factor receptor (G-CSFR) and is a direct target of CEBPA, suggesting a selective pressure for acquisition of these mutations in CEBPAbi AML cells. Interestingly, an additional mutation in G-CSFR pathway (STAT5B N642H) was present in a fifth CEBPAbi AML sample.Considering the high frequency of CSF3R mutations in CEBPAbi AML we conducted a targeted chemical screen employing a collection of compounds (n=11) enriched for JAK inhibitors. Inhibitors were tested in a dose response assays using CEBPAbi (n=14) and control normal karyotype CEBPA wild-type (NK CEBPAwt, n=14) primary AML cells. Results showed that all CSF3R T618I mutated samples were sensitive to JAK inhibitors (e.g. median ruxolitinib IC50 = 66nM (range: 48 - 94)). Most interestingly, CEBPAbi GEP+ samples (n=11), irrespective of their CSF3R mutation status, were uniformly and significantly more sensitive than NK CEBPAwt specimens to JAK inhibitors (e.g. ruxolitinib median IC50: 62 vs 181 nM, p = 0.01), but not to other inhibitors such as dasatinib or sorafenib, or to cytotoxic agents daunorubicin and cytarabine. This may suggest that networks upstream of JAK-STAT are aberrantly activated in a majority of these specimens. In contrast, CEBPAbi GEP- were less sensitive to JAK inhibitors (e.g. ruxolitinib median IC50: 285, range: 48 to >10,000 nM), with the exception of a single sample carrying CSF3R T618I mutation. These results indicate that the transcriptionally distinct CEBPAbi GEP- AML are also distinguishable from CEBPAbi GEP+ AML in their responses to various chemical compounds.
Conclusion
Our study reports a novel co-occurrence of mutations within the CEBPA/CSF3R pathway in CEBPAbi AML and reveals a uniform sensitivity to JAK inhibitors in the transcriptionally uniform CEBPAbi GEP+ AML cells. Altogether, it paves the way to personalized clinical trials repositioning JAK inhibitors for CEBPAbi AML.
Session topic: AML Biology - Novel targeted therapies
Keyword(s): Acute myeloid leukemia, C/EBP, Janus Kinase inhibitor, Mutation
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:15 - 08:30
Location: Hall A3
Background
Acute myeloid leukemias (AML) with CEBPA mutations define a provisional entity in the WHO 2008 classification. Patients with biallelic CEBPA (CEBPAbi) mutations comprising N-terminal frameshift and C-terminal in frame mutations (hereafter termed typical CEBPAbi AML) characteristically present a normal karyotype and have a favorable clinical outcome. In contrast, characteristics of samples with other combinations of mutations (atypical CEBPAbi AML) are less well established.
Aims
Using the RNA-sequencing data from 415 primary AMLs in Leucegene cohort, we aimed to refine the transcriptomic and mutational landscape of 14 CEBPAbi AML samples (7 typical and 7 atypical) present in this collection, and to interrogate the novel mutations identified in this subgroup in a targeted chemical inhibitor screen.
Methods
We performed RNA-sequencing, comparative transcriptomic analysis, mutation detection, cell culture and chemical screening using the methods previously reported (Lavallée et al, Nature Genetics, 2015).
Results
The 7 typical CEBPAbi specimens are best characterized by 95 differentially expressed genes. Using this gene expression profile (GEP), we next performed a principal component analysis and determined that 4/7 atypical CEBPAbi samples clustered with typical CEBPAbi (hereafter termed GEP+), while 3/7 atypical CEBPAbi samples did not (GEP-). Identification of low HOXA9 expression alone was sufficient to discriminate between GEP+ and GEP- atypical CEBPA AML (> 300 fold median HOXA9 expression ratios).In our cohort of CEBPAbi AML we identified 23 mutated genes, including the previously reported mutations in WT1 and GATA2 in 3/14 (21% each). The most frequent mutations in CEBPAbi AML was the activating CSF3R T618I mutation present in 29% (4/14) of this subgroup compared to only 3/401 CSF3R mutated samples in other AML subtypes (p < 0.0001). CSF3R encodes the granulocyte colony stimulating factor receptor (G-CSFR) and is a direct target of CEBPA, suggesting a selective pressure for acquisition of these mutations in CEBPAbi AML cells. Interestingly, an additional mutation in G-CSFR pathway (STAT5B N642H) was present in a fifth CEBPAbi AML sample.Considering the high frequency of CSF3R mutations in CEBPAbi AML we conducted a targeted chemical screen employing a collection of compounds (n=11) enriched for JAK inhibitors. Inhibitors were tested in a dose response assays using CEBPAbi (n=14) and control normal karyotype CEBPA wild-type (NK CEBPAwt, n=14) primary AML cells. Results showed that all CSF3R T618I mutated samples were sensitive to JAK inhibitors (e.g. median ruxolitinib IC50 = 66nM (range: 48 - 94)). Most interestingly, CEBPAbi GEP+ samples (n=11), irrespective of their CSF3R mutation status, were uniformly and significantly more sensitive than NK CEBPAwt specimens to JAK inhibitors (e.g. ruxolitinib median IC50: 62 vs 181 nM, p = 0.01), but not to other inhibitors such as dasatinib or sorafenib, or to cytotoxic agents daunorubicin and cytarabine. This may suggest that networks upstream of JAK-STAT are aberrantly activated in a majority of these specimens. In contrast, CEBPAbi GEP- were less sensitive to JAK inhibitors (e.g. ruxolitinib median IC50: 285, range: 48 to >10,000 nM), with the exception of a single sample carrying CSF3R T618I mutation. These results indicate that the transcriptionally distinct CEBPAbi GEP- AML are also distinguishable from CEBPAbi GEP+ AML in their responses to various chemical compounds.
Conclusion
Our study reports a novel co-occurrence of mutations within the CEBPA/CSF3R pathway in CEBPAbi AML and reveals a uniform sensitivity to JAK inhibitors in the transcriptionally uniform CEBPAbi GEP+ AML cells. Altogether, it paves the way to personalized clinical trials repositioning JAK inhibitors for CEBPAbi AML.
Session topic: AML Biology - Novel targeted therapies
Keyword(s): Acute myeloid leukemia, C/EBP, Janus Kinase inhibitor, Mutation
Abstract: S804
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:15 - 08:30
Location: Hall A3
Background
Acute myeloid leukemias (AML) with CEBPA mutations define a provisional entity in the WHO 2008 classification. Patients with biallelic CEBPA (CEBPAbi) mutations comprising N-terminal frameshift and C-terminal in frame mutations (hereafter termed typical CEBPAbi AML) characteristically present a normal karyotype and have a favorable clinical outcome. In contrast, characteristics of samples with other combinations of mutations (atypical CEBPAbi AML) are less well established.
Aims
Using the RNA-sequencing data from 415 primary AMLs in Leucegene cohort, we aimed to refine the transcriptomic and mutational landscape of 14 CEBPAbi AML samples (7 typical and 7 atypical) present in this collection, and to interrogate the novel mutations identified in this subgroup in a targeted chemical inhibitor screen.
Methods
We performed RNA-sequencing, comparative transcriptomic analysis, mutation detection, cell culture and chemical screening using the methods previously reported (Lavallée et al, Nature Genetics, 2015).
Results
The 7 typical CEBPAbi specimens are best characterized by 95 differentially expressed genes. Using this gene expression profile (GEP), we next performed a principal component analysis and determined that 4/7 atypical CEBPAbi samples clustered with typical CEBPAbi (hereafter termed GEP+), while 3/7 atypical CEBPAbi samples did not (GEP-). Identification of low HOXA9 expression alone was sufficient to discriminate between GEP+ and GEP- atypical CEBPA AML (> 300 fold median HOXA9 expression ratios).In our cohort of CEBPAbi AML we identified 23 mutated genes, including the previously reported mutations in WT1 and GATA2 in 3/14 (21% each). The most frequent mutations in CEBPAbi AML was the activating CSF3R T618I mutation present in 29% (4/14) of this subgroup compared to only 3/401 CSF3R mutated samples in other AML subtypes (p < 0.0001). CSF3R encodes the granulocyte colony stimulating factor receptor (G-CSFR) and is a direct target of CEBPA, suggesting a selective pressure for acquisition of these mutations in CEBPAbi AML cells. Interestingly, an additional mutation in G-CSFR pathway (STAT5B N642H) was present in a fifth CEBPAbi AML sample.Considering the high frequency of CSF3R mutations in CEBPAbi AML we conducted a targeted chemical screen employing a collection of compounds (n=11) enriched for JAK inhibitors. Inhibitors were tested in a dose response assays using CEBPAbi (n=14) and control normal karyotype CEBPA wild-type (NK CEBPAwt, n=14) primary AML cells. Results showed that all CSF3R T618I mutated samples were sensitive to JAK inhibitors (e.g. median ruxolitinib IC50 = 66nM (range: 48 - 94)). Most interestingly, CEBPAbi GEP+ samples (n=11), irrespective of their CSF3R mutation status, were uniformly and significantly more sensitive than NK CEBPAwt specimens to JAK inhibitors (e.g. ruxolitinib median IC50: 62 vs 181 nM, p = 0.01), but not to other inhibitors such as dasatinib or sorafenib, or to cytotoxic agents daunorubicin and cytarabine. This may suggest that networks upstream of JAK-STAT are aberrantly activated in a majority of these specimens. In contrast, CEBPAbi GEP- were less sensitive to JAK inhibitors (e.g. ruxolitinib median IC50: 285, range: 48 to >10,000 nM), with the exception of a single sample carrying CSF3R T618I mutation. These results indicate that the transcriptionally distinct CEBPAbi GEP- AML are also distinguishable from CEBPAbi GEP+ AML in their responses to various chemical compounds.
Conclusion
Our study reports a novel co-occurrence of mutations within the CEBPA/CSF3R pathway in CEBPAbi AML and reveals a uniform sensitivity to JAK inhibitors in the transcriptionally uniform CEBPAbi GEP+ AML cells. Altogether, it paves the way to personalized clinical trials repositioning JAK inhibitors for CEBPAbi AML.
Session topic: AML Biology - Novel targeted therapies
Keyword(s): Acute myeloid leukemia, C/EBP, Janus Kinase inhibitor, Mutation
Type: Oral Presentation
Presentation during EHA21: On Sunday, June 12, 2016 from 08:15 - 08:30
Location: Hall A3
Background
Acute myeloid leukemias (AML) with CEBPA mutations define a provisional entity in the WHO 2008 classification. Patients with biallelic CEBPA (CEBPAbi) mutations comprising N-terminal frameshift and C-terminal in frame mutations (hereafter termed typical CEBPAbi AML) characteristically present a normal karyotype and have a favorable clinical outcome. In contrast, characteristics of samples with other combinations of mutations (atypical CEBPAbi AML) are less well established.
Aims
Using the RNA-sequencing data from 415 primary AMLs in Leucegene cohort, we aimed to refine the transcriptomic and mutational landscape of 14 CEBPAbi AML samples (7 typical and 7 atypical) present in this collection, and to interrogate the novel mutations identified in this subgroup in a targeted chemical inhibitor screen.
Methods
We performed RNA-sequencing, comparative transcriptomic analysis, mutation detection, cell culture and chemical screening using the methods previously reported (Lavallée et al, Nature Genetics, 2015).
Results
The 7 typical CEBPAbi specimens are best characterized by 95 differentially expressed genes. Using this gene expression profile (GEP), we next performed a principal component analysis and determined that 4/7 atypical CEBPAbi samples clustered with typical CEBPAbi (hereafter termed GEP+), while 3/7 atypical CEBPAbi samples did not (GEP-). Identification of low HOXA9 expression alone was sufficient to discriminate between GEP+ and GEP- atypical CEBPA AML (> 300 fold median HOXA9 expression ratios).In our cohort of CEBPAbi AML we identified 23 mutated genes, including the previously reported mutations in WT1 and GATA2 in 3/14 (21% each). The most frequent mutations in CEBPAbi AML was the activating CSF3R T618I mutation present in 29% (4/14) of this subgroup compared to only 3/401 CSF3R mutated samples in other AML subtypes (p < 0.0001). CSF3R encodes the granulocyte colony stimulating factor receptor (G-CSFR) and is a direct target of CEBPA, suggesting a selective pressure for acquisition of these mutations in CEBPAbi AML cells. Interestingly, an additional mutation in G-CSFR pathway (STAT5B N642H) was present in a fifth CEBPAbi AML sample.Considering the high frequency of CSF3R mutations in CEBPAbi AML we conducted a targeted chemical screen employing a collection of compounds (n=11) enriched for JAK inhibitors. Inhibitors were tested in a dose response assays using CEBPAbi (n=14) and control normal karyotype CEBPA wild-type (NK CEBPAwt, n=14) primary AML cells. Results showed that all CSF3R T618I mutated samples were sensitive to JAK inhibitors (e.g. median ruxolitinib IC50 = 66nM (range: 48 - 94)). Most interestingly, CEBPAbi GEP+ samples (n=11), irrespective of their CSF3R mutation status, were uniformly and significantly more sensitive than NK CEBPAwt specimens to JAK inhibitors (e.g. ruxolitinib median IC50: 62 vs 181 nM, p = 0.01), but not to other inhibitors such as dasatinib or sorafenib, or to cytotoxic agents daunorubicin and cytarabine. This may suggest that networks upstream of JAK-STAT are aberrantly activated in a majority of these specimens. In contrast, CEBPAbi GEP- were less sensitive to JAK inhibitors (e.g. ruxolitinib median IC50: 285, range: 48 to >10,000 nM), with the exception of a single sample carrying CSF3R T618I mutation. These results indicate that the transcriptionally distinct CEBPAbi GEP- AML are also distinguishable from CEBPAbi GEP+ AML in their responses to various chemical compounds.
Conclusion
Our study reports a novel co-occurrence of mutations within the CEBPA/CSF3R pathway in CEBPAbi AML and reveals a uniform sensitivity to JAK inhibitors in the transcriptionally uniform CEBPAbi GEP+ AML cells. Altogether, it paves the way to personalized clinical trials repositioning JAK inhibitors for CEBPAbi AML.
Session topic: AML Biology - Novel targeted therapies
Keyword(s): Acute myeloid leukemia, C/EBP, Janus Kinase inhibitor, Mutation
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