Enhanced ability of DT-IL3 to induce apoptosis in oncogene transformed human hematopoietic cells

Leukemic blasts from patients with acute phase chronic myeloid leukemia and refractory acute myeloid leukemia are highly resistant to many cytotoxic drugs. To overcome mult-drug resistance, we engineered a diptheria fusion protein by fusing human interleukin3 to a truncated form of diptheria toxin (DT). This DT-IL3 protein was more cytotoxic to IL-3R bearing human myeloid leukemia cell lines than IL-3R negative cell lines. Furthermore, exposure of monnuclear cells to DT-IL3 reduced the number of cells capable of forming colonies in semi-solid media. The sensitivity to DT-IL3 of leukemic progenitors from a number of acute phase patients suggests that this agent could have therapeutic potential for some patients with CML. To further investigate the ability of DT-IL3 to selectively inhibit the leukemic cell growth, the effects of DT-1L3 on the growth and induction of apoptosis in oncogene-transformed TF-1 human erthroleukemia cells were determined. Treatment of cytokine-dependent parental TF-1 cells with DT-IL3 results in the induction of apoptotic cell death after 2-3 days as determined by DNA laddering and Annexin V/PI binding. TF-1 cells were transformed to independence of exogenous cytokines after transfection with activated signal transduction molecules (Ha-Ras, Src, Raf, MEK1. PI3K. Akt, BCR-ABL and GM-CSF). Interestingly, the cell line which constitutive expresses autocrine GM-CSF as well as the oncogene-transformed cells, remained sensitve to the effects of DT-IL3 which indicated that cells which had by passed IL-3 signal transduction that retained IL-3 receptor expression were still sensitive to DT-IL3. The ICMs for DTIL3 remained relatively constant with less than 3-fold variation. In contrast, the cytokineindependent TF-1 cells were resistant to cell death induced by certain other chemotherapeutic agents such as all Irans retinoic acid (ATRA) as the IC5(J increases from 1 M.M in the cytokine-dependent cells to 50 μM in the oncogene-transformed cells. Similar results were observed with cis RA. Overexpression of the Bcl-2 oncogene in oncogenetransformed cells did not prevent the cells from the apoptotic-inducing effects of DT-IL3. The oncogene and autocrine transformed cell lines formed tumors in immunocompromised mice whereas the parental cytokine-dependent line does not. The ability of DT-II.3 treatment to reduce leukemic tumor load is being determined. These studies indicate DTIL3 may be an effective adjuvant in the treatment of certain leukemias which are resistant to differentiation-inducing agents such as ATRA.