INTEGRATIVE GENOMICS IDENTIFIES THE MOLECULAR BASIS OF RESISTANCE TO AZACITIDINE THERAPY IN MYELODYSPLASTIC SYNDROMES
(Abstract release date: 05/19/16)
EHA Library. Unnikrishnan A. 06/11/16; 135199; S443
Dr. Ashwin Unnikrishnan
Contributions
Contributions
Abstract
Abstract: S443
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Hall C11
Background
Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5’-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk MDS and CMML and response is associated with improved survival benefits. However, only half of treated patients will ever respond to AZA and the molecular basis for poor response is currently unknown. Additionally, AZA response is rarely sustained and a substantial fraction of responders will eventually relapse.
Aims
We aimed to: 1.) understand the molecular basis for poor response to AZA, and 2.) characterise the in vivo effect of AZA therapy on dysplastic cells in responders, as a first step towards understanding eventual relapse.
Methods
We enrolled 18 high-risk MDS and CMML patients on a compassionate access program for AZA in Australia. Bone marrow was collected at seven different points – before treatment; through 6 cycles of treatment; and at up to two years after initiation - and we isolated high-purity CD34+ HSPCs (Figure A). 10 patients had a complete response while 8 were poorer responders. We performed RNA-seq to query the transcriptomes and deduced the clonal evolution in the bone marrow in response to AZA therapy by whole exome-sequencing and single-cell genotyping.
Results
We hypothesised that primary AZA resistance would be driven by pre-existing molecular differences between responders and non-responders. Analysis of the pre-treatment RNA-seq data revealed differential gene expression between responders and non-responders (Figure B). Pathway analyses of these genes indicated that cell cycle was relatively up-regulated in responders compared to non-responders (Figure C). We validated these gene expression differences in independent patient cohorts. We then adapted a flow cytometry based assay, amenable to prospective use in a clinical diagnostic setting, to directly detect the increased quiescence of CD34+ CD38+ haematopoietic progenitors in unsorted bone marrows of non-responders (Figure D). Finally, to reverse the quiescence of progenitor cells of non-responders, we developed a stromal co-culture drug testing platform and discovered that inhibiting integrin-linked signalling combinatorially with AZA improved the functionality of dysplastic cells (Figure E). To trace the fate of dysplastic cells upon AZA therapy, we performed whole exome sequencing of all patients (Figure F). Using the mutations as “molecular barcodes”, we deduced the clonal architecture in each individual. We have discovered that although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders (Figure G). Lastly, we have also discovered that AZA response is associated with an up-regulation of inflammation-associated pathways in vivo.
Conclusion
Our findings, across independent cohorts and relevant to both MDS and CMML, have immediate clinical utility not simply to prospectively identify AZA non-responders but also by suggesting combinatorial therapies that could improve response. Finally, elucidating the in vivo effects of AZA therapy lay the foundation for developing more durable treatments.
Session topic: Myelodysplastic syndromes - Biology
Keyword(s): Chronic myelomonocytic leukemia, Epigenetic, MDS, Myelodysplasia
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Hall C11
Background
Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5’-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk MDS and CMML and response is associated with improved survival benefits. However, only half of treated patients will ever respond to AZA and the molecular basis for poor response is currently unknown. Additionally, AZA response is rarely sustained and a substantial fraction of responders will eventually relapse.
Aims
We aimed to: 1.) understand the molecular basis for poor response to AZA, and 2.) characterise the in vivo effect of AZA therapy on dysplastic cells in responders, as a first step towards understanding eventual relapse.
Methods
We enrolled 18 high-risk MDS and CMML patients on a compassionate access program for AZA in Australia. Bone marrow was collected at seven different points – before treatment; through 6 cycles of treatment; and at up to two years after initiation - and we isolated high-purity CD34+ HSPCs (Figure A). 10 patients had a complete response while 8 were poorer responders. We performed RNA-seq to query the transcriptomes and deduced the clonal evolution in the bone marrow in response to AZA therapy by whole exome-sequencing and single-cell genotyping.
Results
We hypothesised that primary AZA resistance would be driven by pre-existing molecular differences between responders and non-responders. Analysis of the pre-treatment RNA-seq data revealed differential gene expression between responders and non-responders (Figure B). Pathway analyses of these genes indicated that cell cycle was relatively up-regulated in responders compared to non-responders (Figure C). We validated these gene expression differences in independent patient cohorts. We then adapted a flow cytometry based assay, amenable to prospective use in a clinical diagnostic setting, to directly detect the increased quiescence of CD34+ CD38+ haematopoietic progenitors in unsorted bone marrows of non-responders (Figure D). Finally, to reverse the quiescence of progenitor cells of non-responders, we developed a stromal co-culture drug testing platform and discovered that inhibiting integrin-linked signalling combinatorially with AZA improved the functionality of dysplastic cells (Figure E). To trace the fate of dysplastic cells upon AZA therapy, we performed whole exome sequencing of all patients (Figure F). Using the mutations as “molecular barcodes”, we deduced the clonal architecture in each individual. We have discovered that although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders (Figure G). Lastly, we have also discovered that AZA response is associated with an up-regulation of inflammation-associated pathways in vivo.
Conclusion
Our findings, across independent cohorts and relevant to both MDS and CMML, have immediate clinical utility not simply to prospectively identify AZA non-responders but also by suggesting combinatorial therapies that could improve response. Finally, elucidating the in vivo effects of AZA therapy lay the foundation for developing more durable treatments.
Session topic: Myelodysplastic syndromes - Biology
Keyword(s): Chronic myelomonocytic leukemia, Epigenetic, MDS, Myelodysplasia
Abstract: S443
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Hall C11
Background
Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5’-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk MDS and CMML and response is associated with improved survival benefits. However, only half of treated patients will ever respond to AZA and the molecular basis for poor response is currently unknown. Additionally, AZA response is rarely sustained and a substantial fraction of responders will eventually relapse.
Aims
We aimed to: 1.) understand the molecular basis for poor response to AZA, and 2.) characterise the in vivo effect of AZA therapy on dysplastic cells in responders, as a first step towards understanding eventual relapse.
Methods
We enrolled 18 high-risk MDS and CMML patients on a compassionate access program for AZA in Australia. Bone marrow was collected at seven different points – before treatment; through 6 cycles of treatment; and at up to two years after initiation - and we isolated high-purity CD34+ HSPCs (Figure A). 10 patients had a complete response while 8 were poorer responders. We performed RNA-seq to query the transcriptomes and deduced the clonal evolution in the bone marrow in response to AZA therapy by whole exome-sequencing and single-cell genotyping.
Results
We hypothesised that primary AZA resistance would be driven by pre-existing molecular differences between responders and non-responders. Analysis of the pre-treatment RNA-seq data revealed differential gene expression between responders and non-responders (Figure B). Pathway analyses of these genes indicated that cell cycle was relatively up-regulated in responders compared to non-responders (Figure C). We validated these gene expression differences in independent patient cohorts. We then adapted a flow cytometry based assay, amenable to prospective use in a clinical diagnostic setting, to directly detect the increased quiescence of CD34+ CD38+ haematopoietic progenitors in unsorted bone marrows of non-responders (Figure D). Finally, to reverse the quiescence of progenitor cells of non-responders, we developed a stromal co-culture drug testing platform and discovered that inhibiting integrin-linked signalling combinatorially with AZA improved the functionality of dysplastic cells (Figure E). To trace the fate of dysplastic cells upon AZA therapy, we performed whole exome sequencing of all patients (Figure F). Using the mutations as “molecular barcodes”, we deduced the clonal architecture in each individual. We have discovered that although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders (Figure G). Lastly, we have also discovered that AZA response is associated with an up-regulation of inflammation-associated pathways in vivo.
Conclusion
Our findings, across independent cohorts and relevant to both MDS and CMML, have immediate clinical utility not simply to prospectively identify AZA non-responders but also by suggesting combinatorial therapies that could improve response. Finally, elucidating the in vivo effects of AZA therapy lay the foundation for developing more durable treatments.
Session topic: Myelodysplastic syndromes - Biology
Keyword(s): Chronic myelomonocytic leukemia, Epigenetic, MDS, Myelodysplasia
Type: Oral Presentation
Presentation during EHA21: On Saturday, June 11, 2016 from 11:45 - 12:00
Location: Hall C11
Background
Myelodysplastic Syndrome (MDS) and Chronic Myelomonocytic Leukaemia (CMML) are haematological disorders that develop in haematopoietic stem or progenitor cells (HSPCs) and are characterised by ineffective haematopoiesis. 5’-Azacitidine (AZA), a DNA demethylating agent, is the primary drug for the treatment of high-risk MDS and CMML and response is associated with improved survival benefits. However, only half of treated patients will ever respond to AZA and the molecular basis for poor response is currently unknown. Additionally, AZA response is rarely sustained and a substantial fraction of responders will eventually relapse.
Aims
We aimed to: 1.) understand the molecular basis for poor response to AZA, and 2.) characterise the in vivo effect of AZA therapy on dysplastic cells in responders, as a first step towards understanding eventual relapse.
Methods
We enrolled 18 high-risk MDS and CMML patients on a compassionate access program for AZA in Australia. Bone marrow was collected at seven different points – before treatment; through 6 cycles of treatment; and at up to two years after initiation - and we isolated high-purity CD34+ HSPCs (Figure A). 10 patients had a complete response while 8 were poorer responders. We performed RNA-seq to query the transcriptomes and deduced the clonal evolution in the bone marrow in response to AZA therapy by whole exome-sequencing and single-cell genotyping.
Results
We hypothesised that primary AZA resistance would be driven by pre-existing molecular differences between responders and non-responders. Analysis of the pre-treatment RNA-seq data revealed differential gene expression between responders and non-responders (Figure B). Pathway analyses of these genes indicated that cell cycle was relatively up-regulated in responders compared to non-responders (Figure C). We validated these gene expression differences in independent patient cohorts. We then adapted a flow cytometry based assay, amenable to prospective use in a clinical diagnostic setting, to directly detect the increased quiescence of CD34+ CD38+ haematopoietic progenitors in unsorted bone marrows of non-responders (Figure D). Finally, to reverse the quiescence of progenitor cells of non-responders, we developed a stromal co-culture drug testing platform and discovered that inhibiting integrin-linked signalling combinatorially with AZA improved the functionality of dysplastic cells (Figure E). To trace the fate of dysplastic cells upon AZA therapy, we performed whole exome sequencing of all patients (Figure F). Using the mutations as “molecular barcodes”, we deduced the clonal architecture in each individual. We have discovered that although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders (Figure G). Lastly, we have also discovered that AZA response is associated with an up-regulation of inflammation-associated pathways in vivo.
Conclusion
Our findings, across independent cohorts and relevant to both MDS and CMML, have immediate clinical utility not simply to prospectively identify AZA non-responders but also by suggesting combinatorial therapies that could improve response. Finally, elucidating the in vivo effects of AZA therapy lay the foundation for developing more durable treatments.
Session topic: Myelodysplastic syndromes - Biology
Keyword(s): Chronic myelomonocytic leukemia, Epigenetic, MDS, Myelodysplasia
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