Transcriptional Regulators Cooperate to Drive Oncogenic Transcription Networks in High-Risk Acute Myeloid Leukemia

Acute Myeloid Leukemia (AML) is a disease of aged individuals with frequent relapse occurrence. Relapsed and aged AML patients have poor clinical outcomes and are underrepresented in current publicly available patient cohorts. Furthermore, studies regarding AML specimens often lack appropriate control genes and alter gene pathways using modern methods with unknown off-target effects. To address these gaps in AML research, we identified positive control genes for the use of CRISPR/Cas9 gene editing and identified molecular pathways that are recurrently dysregulated in relapsed or aged AML patient cohorts.
In Chapter 2, we utilized CRISPR/Cas9 to disrupt potential factors associated with clinical phenotypes in AML cell models. TP53 is a recurrently mutated or deleted tumor suppressive gene in AML. We demonstrate that TP53 serves as a model positive control to impact cell proliferation. DCK and CDA directly regulate cytarabine efficacy in AML, and we confirmed that knockout of DCK or overexpressing CDA in AML cells increases resistance to chemotherapy. The usage of the methods described in this chapter will better allow scientists to appropriately gauge the biological impact of experimental gene modulation.
In Chapter 3, we identify the LMO2-LDB1-TAL1 complex as a potential regulator of gene transcription changes in AML relapse. TAL1, a master hematopoietic regulator, dysregulates transcription patterns in relapsed AML associated with cell cycle dynamics, heme metabolism, and transcriptional targets of MYC or E2F. TAL1 expression changes may also be indicative of sensitivity to receptor tyrosine kinase inhibitors. However, recurrent changes in TAL1 promoter DNA methylation or gene locus amplification were not observed, suggesting that TAL1 regulation in AML relapse is complex. TAL1 dysregulation in AML cell models disrupts balanced cell growth dynamics and reduces cell growth over time in cell culture models. However, the knockdown of TAL1 did not alter apoptosis or response to the BLC2 inhibitor venetoclax. Furthermore, TAL1 disruption in cell models recapitulated transcriptomic trends observed in AML patients. Chromatin accessibility following TAL1 knockdown in cells identified changes associated with signaling pathways that originate with receptor tyrosine kinases.
In Chapter 4, we determine that the histone methyltransferase COMPASS complex may be responsible for transcription dynamics in both aged and relapsed AML. KMT2C and RBBP5 were independently identified in two different patient cohorts as potentially regulating chromatin and expression dynamics in AML patient specimens. KMT2C mutated or haploid patients exhibited unique transcriptional dysregulation of cell cycle regulation genes, E2F targets, and MYC targets in aged patients. RBBP5 disruption reduces cell growth and proliferation in cell models and may be a critical oncogene in AML relapse.
Collectively, this work highlights that transcriptional regulators in both aged and relapsed AML are potential driving factors behind AML pathogenesis or survival outcomes. Complex protein formation and function is critical for AML cell survival and growth. Disruption of these complexes leads to cell death or reduced growth, indicating potential targetable therapeutic intervention avenues. Future work on these complexes in AML will reveal direct targets, chemotherapeutic vulnerabilities, and exact upstream mechanisms driving dysregulation of these genes in AML patients.