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-INDUCED DIFFERENTIATION AND DIFFERNTIATION RESISTANCE
IN AML CELLS"
Molecular Pathology and Immunology Program
M.Sc. 2004, West China Center of Medical Sciences, Chengdu, China
M.D. 2001, Nanjing Medical University, Nanjing, China
Thesis Advisor: George P. Studzinski, Ph.D, M.D.
Department of Pathology and Laboratory Medicine
Monday, March 26, 2012
1:00 P.M., MSB Room C-555
For three decades 1Š,25-dihydroxyvitamin D3 (1,25D) was known to be able to overcome the block to differentiation of cultured acute myeloid leukemia (AML) cells. However, clinical trials of vitamin D derivatives (VDD) showed unremarkable results, suggesting the development of vitamin D resistance in patientsí AML blasts. MAPK signaling is among the major networks that respond to various environmental stimuli and regulate AML cell survival, proliferation, differentiation and apoptosis. This study was initiated by measuring 1,25D-induced alterations in MAPK expression profile and then focused on HPK1-JNK signaling of differentiation, and differentiation resistance, in subtypes of AML cells. A subline of HL60 cells (40AF cells), which can proliferate in the presence of 1,25D, was used as a model of acquired 1,25D resistance. Such resistance can be partially reversed by the addition of a plant anti-oxidant carnosic acid and a p38MAPK inhibitor SB202190 (DCS cocktail).
MAPK RT2-PCR array was performed to determine mRNA expression of 84 genes in the MAPK network and in cell cycle regulation. In four different subtypes of AML blasts studied ex vivo, 11 genes including HPK1, were up-regulated by VDDs more than 2 fold in at least two patients. Compared to 1,25D-sensitive HL60 cells, 10 genes were up-regulated in 1,25D-resistant 40AF cells; and HPK1 mRNA level increased 54 fold with statistical significance.
Hematopoietic progenitor kinase 1 (HPK1) is a MAP4 Kinase. Although at transcriptional level the expression of HPK1 was high in 1,25D-resistant 40AF cells, this was not responsible for VDD resistance, since knock-down of HPK1 actually reduced 1,25D/DCS-induced differentiation of 1,25D-sensitive HL60 and U937 cells, and of 1,25D-resistant 40AF cells. The activated JNK-cJun-C/EBP‚ pathway, which is related to differentiation, was also inhibited by HPK1 knock-down. The resistance may be due, at least in part, to the increased level of a cleaved fragment of HPK1 (HPK1-C), which is low in sensitive cells but high in both 40AF cells and in the intrinsically vitamin D-resistant KG-1a cells. Consistent with this hypothesis, when differentiation of 40AF or KG-1a cells was induced by DCS, the level of HPK1-C fragment decreased, while the level of full-length (FL)-HPK1 protein increased. Accordingly, inhibition of proteolysis with pan-caspase inhibitor Q-VD-OPh (QVD) reduced HPK1 cleavage and enhanced DCS-induced differentiation in 40AF cells. Also, the inhibition of caspase activity by QVD enhanced differentiation of AML patientsí blasts induced by VDD in an AML subtype/cell context-dependent manner. These results indicate that FL-HPK1 is a positive regulator of vitamin D-induced differentiation in AML cells, while the cleaved HPK1 fragment contributes to vitamin D resistance. Thus, HPK1-JNK signaling and the caspases warrant attention as novel molecular targets in the therapy of leukemia.