L-asparaginase is a bacterial enzyme that depletes serum asparagine to inhibit leukemic cell growth in acute lymphoblastic leukemia. Some patients do not respond to L-asparaginase and the cellular mechanisms underlying non-response remain poorly understood. Under asparagine deprivation, cells activate a transcriptional program known as the amino acid deprivation response that is mediated by activating transcription factor 4 (ATF4). This program culminates in the expression of asparagine synthetase (ASNS), the enzyme responsible for the synthesis of asparagine. Other than ATF4, the transcriptional regulators involved in this response are poorly defined. To identify transcriptional genes involved in the response to L-asparaginase, I developed a CRISPR-Cas9 genetic screening approach to individually knock out each transcription-related gene in the genome. This approach allowed determination of genes that were required for the resistance of an ALL cell line to L- asparaginase. The top-scoring gene in my preliminary genetic screen was a poorly described transcription factor, ZBTB1. Knockout of ZBTB1 sensitized this ALL cell line to treatment with L-asparaginase. Preliminary work suggested that ZBTB1 enriches in the promoter of ASNS to promote transcription. I hypothesize that ZBTB1 and the regulation of ASNS expression may be a suitable drug target in asparaginase resistant leukemia. A lack of insight into the mechanistic role of ZBTB1 in mediating transcription, however, precludes investigation of this hypothesis. In this proposal, building on my preliminary work, I will test the hypothesis that ZBTB1 positively regulates the transcription of ASNS and may be a therapeutic target for L-asparaginase resistant ALLs.
In Aim 1, I will investigate the mechanism by which ZBTB1 regulates the expression of ASNS.
In Aim 2, I will determine whether ZBTB1 knockout sensitizes human ALL cell lines and primary human ALLs to treatment with L- asparaginase in vivo. I anticipate that these studies will determine: 1) the mechanistic role of ZBTB1 in the response to L-asparaginase in leukemic cells and 2) the potential of ZBTB1 as a therapeutic target in L- asparaginase resistant ALL. This work will be completed in the laboratory of Dr. Kivanc Birsoy with the co- advisement of Dr. Robert Roeder at the Rockefeller University. The training plan outlined in this proposal is designed to best prepare me for a career as an independent physician-scientist following residency and fellowship training in hematology and oncology.
The transcriptional mechanisms underlying the cellular response to L-asparaginase in acute lymphoblastic leukemia remain poorly defined. This proposal seeks to (1) gain mechanistic insight into the role of ZBTB1 in the response to L-asparaginase and (2) evaluate ZBTB1 as a therapeutic target for asparaginase-resistant leukemia. Completion of this work will provide insight into the transcriptional mechanisms by which ALLs develop resistance to L-asparaginase.
