Uncovering the Structural Rearrangements in the Philadelphia Chromosome
Introduction
Philadelphia-positive leukemia accounts for over 90% of Chronic Myeloid Leukemia (CML) patients and around 25% of Acute Lymphoblastic Leukemia (ALL). Philadelphia-positive leukemia is characterized by the translocation t(9;22)(q34;q11), which results in the oncogene BCR-ABL1 and a chromosome called the Philadelphia (Ph) chromosome (chr). In adults, Philadelphia-positive leukemia is a poor prognosis marker. While imatinib, Tyrosine Kinase Inhibitor (TKI), is the current front-line treatment for Philadelphia-positive leukemia, 20-30% of patients are initially non-responsive or will eventually gain resistance to the drug. A recent study by Fabian-Morales et al., 2022, linked inherent imatinib resistance to specific genome organization, mainly disordered chromatin domains. Genome organization is a highly complex process by which chromatin is segregated within the nucleus into specific domains, usually segregated by megabases of space and by epigenetic marks, making stable translocation highly unfavorable. How translocations like the Philadelphia chromosome are stabilized has been historically understudied. Here we aim to examine how the Ph chromosome is stabilized through the similarity or difference of compartment states. This study aims to be the first to visualize an entire abnormal chromosome with high spatial and genomic resolution at the single-cell level.
Methods
Using OligoSTORM, a super-resolution microscopy technique, we aim to image the entirety of the Ph chromosome at nanoscale resolution. We designed an oligo library that traces the entire Ph chromosome at 100kb resolution. In addition to the DNA super-resolution trace, we have designed an RNA library to image a series of RNAs from the Ph chromosome on the same sample as the DNA trace, allowing us to connect the transcriptional data to the chromatin structure. We also submitted RNA-sequencing samples to identify any changes at the transcription level.
Results
We have successfully traced portions of the Ph chromosome, with plans to trace it in its entirety in the near future. We have preliminary data suggesting that chr9 and chr22 segregate close to each other in non-leukemia healthy cells. We have also proven that our library design and methodology work to achieve the primary 500kb resolution, with preliminary data showing that the 100kb resolution is also achievable.
Conclusion
Preliminary data suggests that chr9 and chr22 in cells, that don't typically have a Ph chromosome, segregate within proximity to translocate successfully. The data also shows that our library design is capable of reaching nanoscale resolution by using 100kb steps to trace the Ph chromosome.