Overcoming The Differentiation Block: Mechanisms Of Retinoic Acid-Induced Differentiation, Resistance, And Lineage Selection In Myeloid Leukemia Progenitor Cells
Differentiation induction therapy aims to "reform, rather than retaliate" like conventional chemotherapeutic methods. The most successful differentiation therapy has been treatment of t(15;17)-positive acute promyelocytic leukemia (APL) patients with retinoic acid (RA), an embryonic morphogen and derivative of vitamin A. Broad application of RA-induced differentiation therapy is currently limited by naïve/emergent RA resistance, cell-type specific responsivity, and the ambiguous mechanism of RA action. This dissertation provides an introduction to RA differentiation therapy and myeloid leukemia cell lines in Chapter 1. Using patient-derived, HL60 myeloblastic (FAB M2) leukemia cells, where all-trans retinoic acid (RA) induces granulocytic differentiation, we developed two sequentially emergent RA-resistant HL60 cell lines which are characterized by loss of RA-inducible G1/G0 arrest, CD11b expression, oxidative metabolism, and signaling factor expression (reviewed in Chapter 2). All materials and methods are described in Chapter 3. We found that the Src-family kinase inhibitor PP2 can rescue differentiation markers in RA-resistant HL60 cells (Chapter 4). Investigating additional kinase inhibitors targeting an interrelated kinase network revealed that the c-Raf inhibitor GW5074 also rescues RA-induced differentiation, and clarified that a highly correlated, RA-induced c-Raf/Lyn module is uncoupled from traditional downstream events like Akt and ERK activation (Chapter 5). Focusing on the lineage selection aspects of RA (granulocytic) vs. vitamin D3 (monocytic) induction differentiation in bipotent HL60 cells, we discovered that RA-resistant HL60 display a progressive reduced response to D3 treatment (Chapter 6). Finally, we compared phenotype and transcription factor expression in wild-type and RA-resistant HL60 to the myeloid cell lines NB4, U937, and K562, and assessed the fundamental lineage-specific induction mechanisms in cells of varying hematopoietic maturity (Chapter 7). RA-resistant cells provide a means to test combination treatment efficacy, and comparatively can elucidate the crucial mechanisms required for the wild-type response. RA resistance can be combated with kinase inhibitors, which should be selected in-context based on their anti-proliferative and pro-differentiative capability. Another differentiation-inducing agent, vitamin D3, cannot necessarily abrogate temporally segregated early or late RA resistance defect(s); but D3 can induce significant (if not complete) monocytic differentiation in RAresistant cells. Despite divergence of original isolates and limitations of monoculture, a panel of cell lines can clarify fundamental mechanisms and responses to differentiation induction agents. Potential future directions and closing remarks are offered in Chapter 8.
retinoic acid; differentiation; resistance
Varner, Jeffrey D.
Yen, Andrew; Clancy, Paulette
Ph.D. of Chemical Engineering
Doctor of Philosophy
dissertation or thesis