Overexpression of Oncogenic KRAS G12V in Human Stem and Progenitor Cells Enhances Proliferation and Initiates Monocytic Differentiation Via Intrinsic and Extrinsic Pathways.

Abstract 3975

Poster Board III-911

In human hematopoietic malignancies, Ras mutations are frequently present in monocytic and T-cell leukemias. In this study we investigated KRAS G12V-induced phenotypes in human stem and progenitor cells and identified signal transduction pathways that are involved. Using a retroviral expression system, KRAS G12V was introduced to human CD34+ cord blood (CB) cells and proliferation, differentiation and stem cell/progenitor frequencies were evaluated. Overexpression of constitutively active KRAS G12V induced a strong increase in cell expansion over 5-fold in MS5 bone marrow stromal cocultures as well as in cytokine-driven liquid cultures, which coincided with increased early cobblestone formation and induction of monocytic differentiation. Erythroid progenitors were greatly reduced by introduction of KRAS G12V and Q-PCR analysis revealed that expression of PU.1 was increased in conjunction with reduced GATA1 expression in KRAS G12V cells. Progenitor frequencies were increased 6-fold in KRAS-transduced cells within 1 week after plating on MS5. By week three progenitors were exhausted and KRAS-transduced cells were terminally differentiated into monocytes/macrophages. These results were in line with the strong reduction in LTC-IC frequencies at week 5, indicating that also the stem cell pool was exhausted. Intriguingly, when KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, we observed that the non-transduced cells also displayed a strong growth advantage, coinciding with enhanced early cobblestone formation. Furthermore, the addition of conditioned medium from KRAS G12V-transduced cells grown on MS5 to non-transduced CB cells induced a strong growth advantage and formation of early CAFCs. These observations indicate that, besides intrinsic pathways, secreted factor(s) play an important role in the phenotypes induced by KRAS G12V in human CB CD34+ cells. Current studies include mass-spectroscopy analysis of the secretome of KRAS G12V-transduced CB CD34+ cells to identify the factor(s) that are involved.

In order to elucidate signal transduction pathways that mediate KRAS G12V-induced phenotypes, Western-blot analysis was performed. These experiments revealed an increase in phospho-ERK1/2, phospho-p38 and phospho- STAT5 (Y694) levels in KRAS-transduced cells, whereas phospho-JNK was not induced and phospho-C/EBPa (S21) levels were slightly reduced. Induction of STAT5 Y649 phosphorylation by KRAS G12V was confirmed by intracellular phosphoFACS analysis, whereby both in HSCs as well as in more committed MPPs KRAS-induced phosphorylation of STAT5 was observed. KRAS-transduced cells did not show GM-CSF hypersensitivity in any measured cell population upon activation. Inhibition of the ERK/MAPK pathway using the MEK inhibitor U0126 resulted in strongly reduced expansion in MS5 cocultures, whereby both intrinsically induced proliferation as well as proliferation induced via secreted factor(s) were impaired. KRAS G12V-induced monocytic differentiation was not significantly affected by MEK inhibition. While inhibition of the JNK pathway hardly affected proliferation and differentiation of KRAS G12V cells, inhibition of the p38 pathway using SB203580 inhibitor impaired both proliferation and differentiation. When KRAS G12V-transduced cells were cocultured with non-transduced CB CD34+ cells, inhibition of p38 predominantly affected the transduced cells but not the non-transduced cells, suggesting that the p38 pathway particularly mediates intrinsic phenotypes imposed by KRAS G12V.

In conclusion, we show that overexpression of oncogenic KRAS G12V in human CD34+ cells enhances proliferation and initiates monocytic differentiation via intrinsic and extrinsic pathways.