Melat Gebru
Published: 2020
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AML is a heterogeneous disease caused by several mutations and cytogenetic abnormalities affecting differentiation and proliferation of myeloid lineage cells. FLT3 is a receptor tyrosine kinase frequently overexpressed or mutated, and its mutations are associated with poor prognosis in AML. Although aggressive chemotherapy followed by hematopoietic stem cell transplant is the current standard of care, the recent approval of targeted therapies, such as FLT3 targeted drugs, is revolutionizing AML treatment that had remained unchanged since the 1970s. As more targeted therapies become available, a personalized treatment approach where therapies are tailored to patients' mutation profiles will likely replace aggressive chemotherapies that are often associated with treatment-related mortality, especially in older patients. However, despite the dramatic clinical response to targeted agents, such as FLT3 inhibitors, remission is almost invariably short-lived and ensued by relapse and drug resistance. Hence, there is an urgent need to understand the molecular mechanisms underlying drug resistance in order to prevent relapse. The overarching aim of this dissertation was to understand the mechanisms by which FLT3 inhibitor-induced molecular alterations promote cell survival, and to identify drugs that can target those pro-survival changes. Recent accumulating evidence points to the presence of a transitional population of cells between the start of treatment and acquisition of mutational resistance, called drug-tolerant 'persisters' (DTPs). DTPs can tolerate an otherwise cytotoxic dose of targeted drug treatment by re-wiring their signaling pathways in response to therapy. The central hypothesis of this dissertation is that DTPs undergo transcriptomic alterations immediately after FLT3 inhibition which not only allow them to survive treatment, but also cause them to be uniquely vulnerable to new drugs as compared to treatment-naïve cells. In Chapter 2 of this dissertation, we sought to delineate the dynamic transcriptomic state associated with DTPs that survive lethal FLT3 inhibition. Our findings suggested that FLT3 targeted drug treatment induces differential expression of approximately 2000 genes in the remaining DTPs. Genes involved in inflammatory pathways were the most significantly up-regulated in DTPs. While this transcriptomic change likely promotes their survival, it also confers on them susceptibility to anti-inflammatory drugs. Indeed, our drug screen revealed that DTPs are particularly sensitive to glucocorticoids. We used different FLT3 mutant AML cell lines, patient cells as well as mouse models to validate that the combination of FLT3 inhibitors and glucocorticoids induces a synergistic cell death. In-silico prediction of upstream regulators of the up-regulated genes revealed that inflammatory transcription factors, including NF-[kappa]B, STAT3 and CIITA are activated. We confirmed that DTPs have an increased activation of NF-[kappa]B as compared to treatment naïve cells. In Chapter 3 of this dissertation, we dissect the mechanism by which glucocorticoids and FLT3 inhibitors synergize. We found that FLT3 inhibition causes the up-regulation of glucocorticoid receptor making DTPs susceptible to glucocorticoids. Furthermore, we demonstrated that glucocorticoids act through glucocorticoid receptor to increase the expression of the pro-apoptotic protein Bim and up-regulate the degradation of the anti-apoptotic protein Mcl-1. Together, our data demonstrate that disequilibrium between Bim and Mcl-1 is the key mechanism by which the combination of FLT3 inhibitors and glucocorticoids synergize to augment cell death. In summary, in this dissertation, we have uncovered that the combination of FLT3 inhibitors and glucocorticoids is a novel potential treatment strategy that can eliminate or minimize minimal residual disease and thereby prevent relapse in FLT3 mutant AML patients. This has a significant clinical implication because glucocorticoids have been in clinical use for decades, and hence, the proposed combination therapy can be explored and translated rapidly to improve patient outcome.
