Periodic Reporting for period 1 - scGATA2track (Single cell tracking of malignant transformation in GATA2 deficiency)
Período documentado: 2021-09-01 hasta 2023-08-31
Precision medicine has improved overall survival of cancer patients; unfortunately, not all diseases can benefit from this practice due to a scarce understanding of their molecular mechanisms. This is the case of GATA2 deficiency, a complex multi-system disorder characterized by bone marrow failure, immunodeficiency and high risk to develop myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Chemotherapy and allogenic hematopoietic stem cell (HSC) transplantation are the only available treatments, underlining the lack of predictive tools as a clear medical need.
Penetrance and expressivity within GATA2 families is often variable, suggesting that cooperating somatic mutations and epigenetic events are required to trigger the disease. I propose to unravel the molecular mechanisms of malignant progression of GATA2 deficiency by combining multi-OMIC approaches with groundbreaking functional assays in human induced pluripotent stem cells (hiPSCs).
First, to identify pathogenic single nucleotide variations and small insertion-deletions, I have performed an integrative analysis combining whole exome sequencing with DNA methylation on 16 well-annotated GATA2 carriers.
Then, I have modelled the stepwise progression of normal cells to MDS through the sequential introduction of recurrent GATA2 mutations and known second driver mutations in hiPSCs by CRISPR/Cas9 genome editing. The hiPSC-derived hematopoietic progenitors have been used for transcriptomic profiling and the chromatin accessibility profiling is still ongoing to decipher the underlying gene regulatory networks among the different genetic backgrounds.
Objectives:
1. Evaluate the genetic/epigenetic status of GATA2deficient patient samples.
2. Elucidating the functional consequences of recurrent GATA2 itself and driver mutations in human-based disease models.
3. Unraveling the clonal hierarchy of genomic aberration in GATA2 deficiency patients
Penetrance and expressivity within GATA2 families is often variable, suggesting that cooperating somatic mutations and epigenetic events are required to trigger the disease. I propose to unravel the molecular mechanisms of malignant progression of GATA2 deficiency by combining multi-OMIC approaches with groundbreaking functional assays in human induced pluripotent stem cells (hiPSCs).
First, to identify pathogenic single nucleotide variations and small insertion-deletions, I have performed an integrative analysis combining whole exome sequencing with DNA methylation on 16 well-annotated GATA2 carriers.
Then, I have modelled the stepwise progression of normal cells to MDS through the sequential introduction of recurrent GATA2 mutations and known second driver mutations in hiPSCs by CRISPR/Cas9 genome editing. The hiPSC-derived hematopoietic progenitors have been used for transcriptomic profiling and the chromatin accessibility profiling is still ongoing to decipher the underlying gene regulatory networks among the different genetic backgrounds.
Objectives:
1. Evaluate the genetic/epigenetic status of GATA2deficient patient samples.
2. Elucidating the functional consequences of recurrent GATA2 itself and driver mutations in human-based disease models.
3. Unraveling the clonal hierarchy of genomic aberration in GATA2 deficiency patients
The heterogenous clinic manifestation of GATA2 deficient patients is a conundrum, the penetrance of the disease is very variable and even GATA2 carriers from the same family have a diverse symptoms or even some of them are asymptomatic. To understand this unknown aspect of the disease, I studied whether epigenetic deregulation has an impact in the GATA2 deficiency. I did the DNA methylation profiling, identifying a completely novel epigenetic signature of the GATA2 patients.
DNA sample from 17 clinically annotated GATA2-mutated individuals were referred to our laboratory from a Spanish multi-center network. The high-dimensional DNA methylation data visualization showed two major groups, GATA2-mutant carriers clustering tightly together and separately from healthy donors. Nevertheless, the asymptomatic patients P1, P16 and P17 (no apparent disease at date of sample collection at age 6, 75 and 49 years old) and member from the same family, were encompassed to healthy donor group. Notably, we observed a DNA hypermethylation pattern associated to GATA2 affected patients in bone marrow samples. This observation suggests the presence of a likely early aberrant DNA methylation at specific loci in GATA2-mutant carriers that might have a potential prognostic utility in identifying early patients at risk for disease progression. The results are included in a peer reviewed, scientific journal, focused in topics related to experimental and clinical hematology, Haematologica IF:11.04.
The second aim was to model the stepwise progression of normal cells to myelodysplastic syndrome (MDS) through the sequential introduction of first germline mutation in the GATA2 gene and after known second driver mutations such as SETBP1 (MDS) and ASXL1 (MDS/AML) by CRISPR/Cas9-mediated genome editing. Taking advantage of an in vitro blood differentiation protocol established in the host laboratory, I evaluated the impact of each mutation using the blood differentiation as a read-out. I have observed that the acquisition of three mutations in GATA2, SETBP1 and ASXL1, decreases the hematopoietic progenitors. This result is related with the clinical manifestation in GATA2 deficient patients, who presented aplasia.
Additionally, I have contributed in the writing of a review about GATA2 deficiency titled GATA2 deficiency and MDS/AML: Experimental strategies for disease modelling and future therapeutic prospects, published peer reviewed, medical journal focusing on hematology and other blood-related topics, British Journal of Haematology, IF: 8.6.
Regarding the dissemination plan, I have participated twice in the seminar of program of regenerative medicine (months 4 and 14); I have attended to the international meetings, EWOG-MDS/SAA conference (October ´21), EHA, (June ´22), the ASEICA conference with a poster presentation November´22 and I attended to the kick-off ERAPerMed meeting in Barcelona (June´22). The exploitation of this proposal was proposed in the annex1; however, due of the interruption of the fellowship, a forthcoming discussion about the intellectual property related issues with IDIBELL is planned.
DNA sample from 17 clinically annotated GATA2-mutated individuals were referred to our laboratory from a Spanish multi-center network. The high-dimensional DNA methylation data visualization showed two major groups, GATA2-mutant carriers clustering tightly together and separately from healthy donors. Nevertheless, the asymptomatic patients P1, P16 and P17 (no apparent disease at date of sample collection at age 6, 75 and 49 years old) and member from the same family, were encompassed to healthy donor group. Notably, we observed a DNA hypermethylation pattern associated to GATA2 affected patients in bone marrow samples. This observation suggests the presence of a likely early aberrant DNA methylation at specific loci in GATA2-mutant carriers that might have a potential prognostic utility in identifying early patients at risk for disease progression. The results are included in a peer reviewed, scientific journal, focused in topics related to experimental and clinical hematology, Haematologica IF:11.04.
The second aim was to model the stepwise progression of normal cells to myelodysplastic syndrome (MDS) through the sequential introduction of first germline mutation in the GATA2 gene and after known second driver mutations such as SETBP1 (MDS) and ASXL1 (MDS/AML) by CRISPR/Cas9-mediated genome editing. Taking advantage of an in vitro blood differentiation protocol established in the host laboratory, I evaluated the impact of each mutation using the blood differentiation as a read-out. I have observed that the acquisition of three mutations in GATA2, SETBP1 and ASXL1, decreases the hematopoietic progenitors. This result is related with the clinical manifestation in GATA2 deficient patients, who presented aplasia.
Additionally, I have contributed in the writing of a review about GATA2 deficiency titled GATA2 deficiency and MDS/AML: Experimental strategies for disease modelling and future therapeutic prospects, published peer reviewed, medical journal focusing on hematology and other blood-related topics, British Journal of Haematology, IF: 8.6.
Regarding the dissemination plan, I have participated twice in the seminar of program of regenerative medicine (months 4 and 14); I have attended to the international meetings, EWOG-MDS/SAA conference (October ´21), EHA, (June ´22), the ASEICA conference with a poster presentation November´22 and I attended to the kick-off ERAPerMed meeting in Barcelona (June´22). The exploitation of this proposal was proposed in the annex1; however, due of the interruption of the fellowship, a forthcoming discussion about the intellectual property related issues with IDIBELL is planned.
The progress beyond the state-of-the art is the identification of an aberrant DNA hypermethylated signature in GATA2 deficiency. Specifically, I described the presence of a subset of aberrant hypermethylated set of genes present in GATA2-mutant carriers at early (and not yet symptomatic) disease stage, which could be potentially used as predictors of clonal evolution. In this context, the implementation of customized methylation-specific assays might be instrumental to validate our findings in larger cohorts of patients and to test its clinical prognostic utility. Finally, a collaborative effort will be essential to increase the number patients with this rare yet high-risk MDS/AML predisposition syndrome, allowing for comprehensive genetic and epigenetic analyses to understand the impact of the secondary hits and/or aberrant DNA methylation on the disease progression.
The second aim was to characterized the impact of the somatic mutation, SETBP1 (S) and ASXL1(A) in cells carrying a GATA2(G) mutations. As I described in the previous section, I observed that the acquisition of the three mutations decreases the hematopoietic progenitors CD34+, CD33+ and CD43+). To understand in detailed the molecular mechanism, we decided to study the transcriptional regulation of the blood progenitors cells. The target population (CD34+, CD33+ and CD43+) is very rare, less than 5% from the total, I have isolated 200 (CD34+, CD33+ and CD43+) cells from each condition (Parental, G, GS and GSA) to perform transcriptomic profiling by mini-bulk RNAseq, I received the sequencing data mid February ´23 and the analysis will be done by the bioinformatician of the host laboratory.
The data coming from the second aim will be discussed with the host laboratory to disseminate it in national/international conferences and make it publicly available through research articles.
The second aim was to characterized the impact of the somatic mutation, SETBP1 (S) and ASXL1(A) in cells carrying a GATA2(G) mutations. As I described in the previous section, I observed that the acquisition of the three mutations decreases the hematopoietic progenitors CD34+, CD33+ and CD43+). To understand in detailed the molecular mechanism, we decided to study the transcriptional regulation of the blood progenitors cells. The target population (CD34+, CD33+ and CD43+) is very rare, less than 5% from the total, I have isolated 200 (CD34+, CD33+ and CD43+) cells from each condition (Parental, G, GS and GSA) to perform transcriptomic profiling by mini-bulk RNAseq, I received the sequencing data mid February ´23 and the analysis will be done by the bioinformatician of the host laboratory.
The data coming from the second aim will be discussed with the host laboratory to disseminate it in national/international conferences and make it publicly available through research articles.