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

Abstract Objective A truncated common β chain (Δβ C ) of the interleukin-3 (IL-3) receptor complex was previously identified as a key factor in inducing autonomous growth of IL-3–independent mutants. Expression of Δβ C in IL-3–dependent hematopoietic cells does not result in immediate factor-independent growth, but increases the frequency of obtaining autonomous mutants by three to four orders of magnitude. This study was designed to delineate the mechanisms by which Δβ C increases the frequency to autonomous growth. Design and Methods Retroviral vectors were used to express Δβ C into IL-3–dependent myeloid cells, which were then tested for factor-independent growth. To determine if secondary genetic events were required for conversion to autonomous growth, elements of the Cre-loxP recombinant system were used to excise Δβ C in factor-independent clones. Results Excision of Δβ C in factor-independent clones revealed two types of phenotypes: reversion to factor-dependent growth (1/8) or continued IL-3–dependent growth (7/8). Analysis of cells that remained factor independent revealed constitutive activation of STAT5, not observed in factor-dependent revertants. Analysis of revertant cells demonstrated the presence of interacting secondary mutations that synergize with Δβ C -induced proliferation. A cysteine residue within the truncated extracellular domain of Δβ C was also found to be required for its oncogenic potential, supporting a model of dimerization for receptor activation. Conclusions The high incidence of obtaining factor-independent mutants from cells expressing Δ β C results from the selection of mutations that either complement Δ β C expression to promote proliferation or that singly or in synergy with other secondary mutations negate the requirement of Δ β C expression for proliferation.