SELECTION OF MYELOID PROGENITORS LACKING BCR ABL MESSENGER-RNA IN CHRONIC MYELOGENOUS LEUKEMIA PATIENTS AFTER IN-VITRO TREATMENT WITH THE TYROSINE KINASE INHIBITOR GENISTEIN/

Chronic myelogenous leukemia (CML) is a clonal disorder of the hematop oietic stem cell characterized by a chimeric BCR/ABL gene giving rise to a 210-kD fusion protein with dysregulated tyrosine kinase activity. We investigated the effect of genistein, a protein tyrosine kinase in hibitor, on the in vitro growth of CML and normal marrow-derived multi potent (colony-forming unit-mix [CFU-Mix]), erythroid (burst-forming u nit-erythroid [BFU-E]), and granulocyte-macrophage (colony-forming uni t-granulocyte-macrophage [CFU-GM]) hematopoietic progenitors. Continuo us exposure of CML and normal marrow to genistein induced a statistica lly significant and dose-dependent suppression of colony formation. Ge nistein doses causing 50% inhibition of CML and normal progenitors wer e not significantly different for CFU-Mix (27 mu mol/L v 23 mu mol/L), BFU-E (31 mu mol/L v 29 mu mol/L), and CFU-GM (40 mu mol/L v 32 mu mo l/L). Preincubation of CML and normal marrow with genistein (200 mu mo l/L for 1 to 18 hours) induced a time-dependent suppression of progeni tor cell growth, while sparing a substantial proportion of long-term c ulture-initiating cells (LTC-IC) from CML (range, 91% +/- 9% to 32% +/ - 3%) and normal marrow (range, 85% +/- 8% to 38% +/- 9%). Analysis of individual CML colonies for the presence of the hybrid BCR/ABL mRNA b y reverse transcription-polymerase chain reaction (RT-PCR) showed that genistein treatment significantly reduced the mean +/- SD percentage of marrow BCR/ABL(+) progenitors both by continuous exposure (76% +/- 18% v 24% +/- 12%, P less than or equal to .004) or preincubation (75% +/- 16% v 21% +/- 10%, P less than or equal to .002) experiments. Pre incubation with genistein reduced the percentage of leukemic LTC-IC fr om 87% +/- 12% to 37% +/- 12% (P less than or equal to .003). Analysis of individual colonies by cytogenetics and RT-PCR confirmed that geni stein-induced increase in the percentage of nonleukemic progenitors wa s not due to suppression of BCR/ABL trascription. Analysis of nuclear DNA fragmentation by DNA gel electrophoresis and terminal deoxynucleot idyl transferase assay showed that preincubation of CML mononuclear an d CD34(+) cells with genistein induced significant evidence of apoptos is. These observations show that genistein is capable of (1) exerting a strong antiproliferative effect on CFU-Mix, BFU-E, and CFU-GM while sparing the more primitive LTC-IC and (2) selecting benign hematopoiet ic progenitors from CML marrow, probably through an apoptotic mechanis m. (C) 1996 by The American Society of Hematology.