Investigation of New Therapeutic Compounds for Juvenile Myelomonocytic Leukemia Using Induced Pluripotent Stem Cells with Stably Activated Ras Pathway

Juvenile myelomonocytic leukemia (JMML) is a chronic, poor prognostic myeloid neoplasm of childhood that is characterized by malignant expansion of monocytic cells. Chemo- and radiotherapy are not effective in JMML, therefore allogeneic hematopoietic stem cell transplantation is the only therapy option for most affected children. Relapse is the most frequent cause of treatment failure and event-free-survival at five years is low (approximately 50%). Recent studies showed that in 90% of JMML patients the proliferation of monocytic tumor cells is driven by mutations in a confined set of genes (KRAS, NRAS, PTPN11, NF1 or CBL) that activate the RAS signalling pathway. Drugs specifically targeting this pathway are therefore attractive candidates for therapy of JMML patients.

As in vitro models of JMML, we generated inducible pluripotent stem cells (iPSC) stably expressing wildtype or activating oncogenic versions of KRAS (G12D) or NRAS (G13D) as well as iPSCs with CRISPR interference mediated downregulated NF1 expression. Manipulation of KRAS, NRAS, and NF1 expression and activation of downstream signaling targets (MEK, ERK) of the Ras pathway were confirmed by RT-PCR and western blot analyses, respectively. After transduction iPSCs retained typical pluripotency markers and could be differentiated into CD34+ and CD45+ cells of the hematopoietic lineage. We then carried out a screen to test the response of these iPSC cell lines to experimental and clinical drugs targeting the Ras signaling pathway, as well as to other compounds suggested to be promising candidate drugs or drugs already in clinical trial for JMML. In our screen the model cell lines were resistant to all tested MEK-inhibitors, including Selumetinib and Trametinib. The broad receptor tyrosine kinase inhibitor Dovitinib and the DNA methyltransferase inhibitor Azacytidine elicited strong responses in all iPSC cell lines regardless of their KRAS, NRAS or NF1 state. This underlines their extensive, but non-targeted killing potential.

In our screen, an experimental small molecule drug induced significantly more cell death in KRAS-G12D iPSCs (IC₅₀ 1.5 µM) than in comparable wildtype cells (IC₅₀ 3.3 µM, p<0.0001), which could be validated in independent assays. In addition to targeted cell death activation, the drug has been suggested to promote differentiation of hematopoietic cells, which could potentially increase its anti-tumor efficiency. Experimental studies analyzing the underlying mechanism of its differential effect on KRAS wildtype compared to KRAS-G12D cells are currently carried out and will be presented.

Our results suggest, that iPSCs with RAS pathway activation due to stable expression of oncogenic KRAS or NRAS or downregulation of NF1 expression are valuable tools for preclinical testing and may identify promising novel lead compounds for JMML treatment.