Functional identification of secondary mutations inducing autonomous growth in synergy with a truncated interleukin-3 receptor: Implications for multi-step oncogenesis

Objective. A truncated common beta chain (Delta betac) of the interleukin-3 (IL-3) receptor complex was previously identified as a key factor in induc ing autonomous growth of IL-3-independent mutants. Expression of Delta beta (C) in IL-3-dependent hematopoietic cells does not result in immediate fac tor-independent growth, but increases the frequency of obtaining autonomous mutants by three to four orders of magnitude, This study was designed to d elineate the mechanisms by which Delta beta (C) increases the frequency to autonomous growth, Design and Methods, Retroviral vectors were used to express Delta beta (C) into IL-3-dependent myeloid cells, which were then tested for factor-indepe ndent growth, To determine if secondary genetic events were required for co nversion to autonomous growth, elements of the Cre-loxP recombinant system were used to excise Delta beta (C) in factor-independent clones, Results. Excision of Delta beta (C) in factor-independent clones revealed t wo types of phenotypes: reversion to factor-dependent growth (1/8) or conti nued IL-3-dependent growth (7/8), Analysis of cells that remained factor in dependent revealed constitutive activation of STAT5, not observed in factor -dependent revertants. Analysis of revertant cells demonstrated the presenc e of interacting secondary mutations that synergize with Delta beta (C)-ind uced proliferation, A cysteine residue within the truncated extracellular d omain of Delta beta (C) was also found to be required for its oncogenic pot ential, supporting a model of dimerization for receptor activation, Conclusions. The high incidence of obtaining factor-independent mutants fro m cells expressing Delta beta (C) results from the selection of mutations t hat either complement Delta beta (C) expression to promote proliferation or that singly or in synergy with other secondary mutations negate the requir ement of Delta beta (C) expression for proliferation. (C) 2001 Internationa l Society for Experimental Hematology. Published by Elsevier Science Inc.