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Klco Lab: Projects

Genomics of Pediatric Myeloid Tumors

Our lab relies heavily on Next Generation Sequencing (NGS) approaches to define the genomic landscape of different myeloid tumors that occur in children. In 2016 we published the most comprehensive study on pediatric MDS involving 46 children with primary pediatric MDS. We discovered that activating mutations in the Ras/MAPK pathway and chromosome 7 deletions (monosomy 7) are the most common somatic alterations. In addition, we described germline mutations in SAMD9 and SAMDL in nearly 20% of children with MDS. We are currently working to define the mutations that drive therapy-related myeloid neoplasms and relapsed AML in children, in particular through our involvement in numerous AML clinical trials at St Jude. The goal of these studies is to define new genomic subgroups of pediatric myeloid tumors and to characterize molecular alterations associated with disease progression and outcome.

Association of different mutations and cytogenetic groups in pediatric MDS (from Schwartz et al, Nat. Commun., 2017).

Clonal evolution of myeloid tumors

We are interested in deciphering the clonal architecture and progression of myeloid tumors from diagnosis to relapse and in response to different chemotherapeutic pressures. These studies harness single cell technologies, including both single cell DNA-seq and single cell RNA-seq.

Left, tSNE analysis of 10X scRNA-seq Genomics data from a pediatric AML sample at diagnosis and relapse. Right, the diagnostic leukemia expressed both myeloid and T antigens by flow cytometry and confirmed by scRNA-seq (CD33 and CD3E plots). The relapse leukemia had more myeloid features, as shown by ELANE expression, and increased transcript levels of the anti-apoptotic gene, IFI6. Lower, Pediatric AML samples harboring a NUP98-NSD1 fusion were interrogated at diagnosis and relapse using the Mission Bio Tapestri platform and their AML panel. There is a clear acquisition of a relapse-enriched WT1 p.R445P mutation and an additional clone with a homozygous p. A387fs in WT1.

In addition, we use ultra-deep sequencing to follow the clearance of somatic mutations after chemotherapy with the goal of establishing new benchmarks of minimal/measurable residual disease in acute leukemias. This work is done in collaboration with Xiaotu Ma in the Department of Computational Biology.

SAMD9 and SAMD9L in Pediatric MDS

SAMD9 and SAMD9L are two interferon inducible genes located on human chromosome 7 at band 7q21. Although the function of these proteins is unknown, heterozygous germline missense mutations predispose children to a variety of diseases, including MDS and AML. These mutations result in a profound inhibition of cell growth leading to the outgrowth of cells that lack the mutation through somatic reversion. This occurs through multiple mechanisms, including somatic copy neutral loss of heterozygosity (CN-LOH), acquisition of mutations in cis that abrogate the germline mutation or deletion of chromosome 7. In addition to removing the pathogenic SAMD9 or SAMD9L germline mutation, deletions of chromosome 7 also results in haploinsufficiency of a number of critical genes and patients can progress to MDS/AML when additional cooperating mutations (e.g. KRAS, SETBP1) are acquired. We are using a combination in vitro and biochemical assays and different model systems, including genetically engineered mouse models, to determine the role of SAMD9/SAMD9L in hematopoiesis and to determine how mutations in these genes ultimately lead to myeloid neoplasms in children.

Generating human model systems for functional and mechanistic studies to evaluate molecular lesions in AML

We are currently using multiple human cell systems to understand the molecular mechanisms of transformation by different genetic alterations common in children. In particular, we use cord blood CD34 cells coupled with lentiviral transductions to introduce mutations and fusions (in particular NUP98 fusions) common in pediatric MDS/AML to determine their impact on cell growth, differentiation and transcriptional networks. We are also using CRISPR-Cas9 approaches to introduce loss of function mutations. In addition to these in vitro approaches, we use xenotransplantation into immunodeficient mice to further investigate the mechanisms of transformation and to establish models for pre-clinical testing. This includes a cohort of patient derived AML xenografts.