Enhanced Selective Inhibition of KRAS Mutant Hematopoietic Progenitor Cell Expansion By MEK and Bcl-2 Inhibition
Authors: Lin Huan-Ting , Kubara Kenji , Yamazaki Kazuto , Takagi Masatoshi , Naruto Takuya , Morio Tomohiro , Okumura Takashi , Otsu Makoto 
Published: October 2019

Abstract
Amidst the complex genetic landscape of cancer, the mutagenic and oncogenic effects caused by RAS mutations alone can be overlooked particularly in the context of hematopoietic malignancies. Therefore identifying drugs that could modulate aberrant RAS-MAPK signaling is clinically important. Here we demonstrate the utilization of an iPS cell-based system that could be adapted for compound screening to identify inhibitors with selective inhibition activity against the expansion of KRAS-mutant hematopoietic progenitor cells (HPCs).
RAS-associated autoimmune lymphoproliferative disorder (RALD) is a leukemia-like disease caused by a single acquired RAS mutation. In this study, BM CD34+ cells from RALD patients were reprogrammed into iPS cells. KRAS-mutant cells could be obtained without necessitating the use of gene editing techniques. Non-mutant cells were used as isogenic controls thus permitting the assessment of unwanted toxicity against healthy cells. Constitutive activation of the RAS-MAPK pathway was confirmed by western blotting in mutant iPS cells. As expected, whole exome sequencing (WES) showed that mutant KRAS (G13C) was the only mutation present in these cells. Therefore, the inherent characteristics of mutant cell expansion can be attributed to this mutation only.
Previously we reported that oncogenic KRAS enforces retention of self-renewal in human iPS cells (Kubara et al. Stem Cell Reports. 2018) due to constitutive activation of the KRAS-MAPK signaling pathway. Notably mutant iPS cells retained pluripotency marker expression even in the absence of bFGF, but not isogenic control cells. When we tested compounds with different mechanisms of action, direct MEK inhibition was the most effective in triggering a loss of pluripotency marker expression in mutant iPS cells in the absence of bFGF. This method permits rapid determination of compounds that could modulate aberrant KRAS-MAPK signaling. However, further verification is required to determine whether inhibitory effects on mutant HPC expansion could also be observed.
Next, differentiated multi-potent (Lin-34+43+) HPCs from iPS cells were expanded in "standard" ex vivo culture conditions using SCF, TPO Flt3L and IL-3. This favored the expansion of control cells. We also found "selective" culture conditions that could selectively expand mutant HPCs. RNA-seq analysis on pre-expanded and expanded HPCs revealed the unique molecular signature of expanding mutant cells, meaning genes that showed upregulation in both "standard" and "selective" culture conditions. There was upregulation of CCND1 and CDK4 in mutant HPCs, which facilitates progression to the S phase of the cell cycle. This was also accompanied by upregulation of p16INK4a and p21Cip1, which is characteristic of senescent cells. Indeed, it could be shown that expanded mutant HPCs acquired senescence features such as increased p16 INK4a and Bcl-xL expression at the protein level and increased SA-b-gal activity compared with control cells. Gene correction of KRAS (G13C) using CRISPR/Cas9 reversed these phenotypes. Therefore CCND1/CDK4 and Bcl-2 may be potential inhibitory targets.
We developed a high-throughput screening method whereby differentiated HPCs were sorted directly into 96-well plates and following expansion, cell numbers were quantified by luminescence detection of ATP. First we screened drugs for selective activity (IC50 value for mutant cells lower than control cells). Consistent with the iPS cell pluripotency assay, we identified Trametinib (MEK), as having selective effects against mutant HPCs in addition to Palbociclib (CCND1/CDK4) and Navitoclax (Bcl-2). When used in combination, the selective inhibitory effect was enhanced without compromising the viability of control cells.
To test these drugs in an in vivo setting, highly purified transduced (KRAS or control) mouse HSCs (Lin-CD34-KSL) were transplanted in a competitive repopulation assay. Recipient mice were given Trametinib and Navitoclax. In the KRAS group, donor cells contributed to a lower level of chimerism. We also developed the use of droplet digital PCR (ddPCR) to monitor the percentage of mutant cells within bulk BM CD34+ cells (mutant and isogenic control) when cultured in the presence of inhibitors. Taken together, our study offers novel insights into mutant KRAS-driven HPC expansion and demonstrate an efficient platform for drug screening.

Disclosures
Kubara: Eisai Co., Ltd.: Employment. Yamazaki:Eisai Co., Ltd.: Employment.