Orthogonal Comparison of Nuclear and Mitochondrial Clonal Architectures in Hematologic Malignancies

Nehali Shah (Advisor: Koichi Takahashi, PhD)

Background:
Acute myeloid leukemia (AML) is a hematologic malignancy characterized by accumulation of mutations that disrupt hematopoietic differentiation and promote clonal expansion. Understanding how these mutations arise and evolve is essential for improving diagnosis, prognosis, and treatment stratification. Current methods are limited by either restricted genomic coverage (targeted panels) or low throughput and high cost (in single-cell whole-genome sequencing, scWGS). An emerging alternative is the use of mitochondrial DNA (mtDNA) mutations as clonal markers.

Objective:
This study aims to determine whether mitochondrial-derived clonal architectures correlate with nuclear-derived clonal architectures in AML, thereby evaluating mtDNA as a scalable orthogonal tool for lineage reconstruction.

Methods:
Bone marrow and peripheral blood samples were collected from four AML patients. Mononuclear cells were isolated and processed using a custom Mission Bio Tapestri single-cell DNA panel to simultaneously capture nuclear driver mutations and full-length mitochondrial variants. Single-cell barcoding was performed, followed by library preparation and sequencing. Data were processed through the Tapestri Pipeline and analyzed using R and Python for quality filtering, UMAP visualization, and clonality inference. Mitochondrial variants were annotated to determine functional impact.
In parallel, scWGS data from previously profiled multiple myeloma patients in remissions samples were analyzed to reconstruct nuclear and mitochondrial phylogenies. The concordance between nuclear and mtDNA-based clades was quantified using Jaccard indices and phylogenetic overlap metrics.

Results:
In the Tapestri AML dataset, distinct subclones defined by nuclear mutations such as DNMT3A, IDH1, and ASXL1 were identified, some of which carried specific heteroplasmic mtDNA variants. However, the correlation between mtDNA and nuclear genotypes was variable - certain clones exhibited co-segregating mtDNA mutations, while others did not. In the scWGS dataset, nuclear and mtDNA-based trees showed partial overlap, particularly in recently expanded lineages, suggesting that mtDNA may capture short-term clonal dynamics but incompletely reflect long-term phylogenies.

Conclusions:

These results indicate that mitochondrial DNA variants can associate with, but do not fully recapitulate, nuclear-derived clonal structures in hematologic malignancies. Integrating mtDNA profiling with single-cell nuclear sequencing provides a complementary and scalable framework for exploring clonal evolution. Future studies are necessary to understand why certain clones carry mtDNA mutations but others do not. They can also investigate whether specific biological or metabolic factors influence which clones acquire mtDNA mutations, refining the use of mtDNA as a lineage-tracing marker in leukemia.

Advisory Committee:

• Koichi Takahashi, PhD, Chair
• Simona Colla, PhD
• Andrew Futreal PhD
• Angela Ting, PhD
• Nicholas Short, PhD