Document Type


Degree Name



Cancer Biology


Hematologic malignancies are predominantly caused by genetic aberrations resulting in the unregulated proliferation or blocked differentiation of hematopoietic progenitors. The identification and characterization of growth-activating mutations, or driver mutations, has resulted in the development of targeted therapeutics that dramatically improve patient survival and decrease treatment-related toxicity. However, the most common types of leukemia lack a recurrent and defining driver mutation, meaning that the continued development of targeted therapies will require the characterization of novel and rare leukemic variants. The most common assay used to characterize the functional impact of novel mutations found in leukemia is the in vitro Ba/F3 transformation assay, which was used in the work described herein to screen primary CSF2RB mutations found across all leukemia subtypes. While characterizing one activating mutation it was discovered that acquired mutations in the transgene could arise in the Ba/F3 assay, potentially resulting in false-positive data. This observation was investigated in detail and recommendations stemming from this inquiry are also provided.

In this dissertation, the discovery of the first CSF2RB-activating human variant found in the germline of a pediatric T-cell acute lymphoblastic leukemia patient is presented. In vitro, CSF2RB R461C transforms Ba/F3 cells to factor-independent growth through ligand-independent activation and stabilization of CSF2RB. CSF2RB R461C increases downstream activation of JAK2/STAT-5, and inhibition of JAK2 results in apoptosis, making this a potentially actionable clinical variant.

During the course of characterizing CSF2RB R461C, the frequent trend for Ba/F3 cells to acquire additional mutations in CSF2RB during the factor withdrawal assay was noted. This observation was validated by additional experiments including other receptor activating mutations. Failure to account for these acquired mutations in the Ba/F3 assay could affect the efficacy of screening for functional mutations in leukemia, resulting in wasted researcher time and false positives.

To date, acquired mutations are largely confined to weak transforming oncogenes, as determined by limiting dilution analysis of Ba/F3 cells. Based upon insights from these studies, the Ba/F3 transformation assay would be substantially improved with two recommendations. First, every transformed Ba/F3 cell line should be sequenced for the full length of the transgene of interest. Second, researchers should adopt limiting dilution assays of Ba/F3 cells when characterizing transforming mutations and report rates of transformation. It is unclear at this time what relevance a mutation’s rate of transformation has on disease, but that question can only be addressed by additional testing of a wide range of mutations.

The research in this dissertation characterizes a rare variant in leukemia and recommends improvements for the most common model system used to screen novel mutations. The investigation of rare mutations in leukemia is an essential step in the pursuit of 3 individualized and targeted therapies. Recommendations provided herein will improve future studies of functional leukemic mutations, ensuring reported results are useful and reproducible.




School of Medicine



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