Akt Activation Is Important In KRAS-Mediated Multistep Leukemogenesis
Authors: Ward Ashley F. , Shieh Angell , Harding-Theobald Emily Rose , Bollag Gideon , Shannon Kevin 
Published: October 2019

Abstract 4200

Activating mutations of Ras are found in approximately 30% of all human malignancies, and 85% of these mutations are in the K-ras isoform. These mutations dysregulate cell cycle progression, proliferation and apoptosis, and have been implicated in both initiation and maintenance of tumors. While mutant K-ras thus represents an attractive therapeutic target, attempts to develop a Ras inhibitor have been unsuccessful to date. K-Ras has three canonical downstream effector pathways: MEK/Erk, PI3K/Akt, and Ral. One or more of these pathways may represent an alternative drug target for Ras-driven malignancies, but it is not yet understood how each of these pathways contributes to leukemogenesis. Partial loss-of-function Ras mutations have been identified that render oncogenic K-Ras (K-RasG12D) unable to interact with one or more downstream effectors. Our lab has previously demonstrated that one such partial loss-of-function mutation, K-RasG12D,Y64G, does not activate the PI3K/Akt pathway. Mice transplanted with hematopoietic progenitor cells transduced with MSCV vectors encoding K-RasG12D,Y64G develop an aggressive T-lineage acute lymphoblastic leukemia (T-ALL) with a median survival of 112 days (Shieh and Shannon, Blood (ASH Annual Meeting Abstracts) 2007 110: Abstract 1617).

To determine if the “missing” Ras effector pathway is deregulated during multistep tumorigenesis, we generated cell lines from K-RasG12D,Y64G leukemias (n=6). Western blot analysis revealed low Ras levels and absent PTEN protein expression in 5 of 6 K-RasG12D,Y64G leukemia cell lines. Quantitative PCR analysis revealed reduced PTEN mRNA levels in these cell lines, which was not due to somatic Pten mutations. As expected, cell lines without detectable PTEN showed high pAkt levels that persisted during serum and cytokine deprivation. One K-RasG12D,Y64G leukemia cell line was remarkable because it contained high levels of Ras and retained PTEN expression. DNA sequence analysis of this cell line unexpectedly revealed both the Y64G substitution and a de novo in frame insertion of two amino acids (arginine and aspartic acid) within the switch II domain of K-Ras, between codons 69 and 70. This insertion was also identified in the primary T-ALL. Murine fetal liver cells engineered to express K-RasG12D,Y64G, 69RN70 induced a dramatic pattern of hypersensitive progenitor growth characterized by cytokine-independent colony formation and large and aberrant CFU-GM morphology in the presence of GM-CSF that was indistinguishable from cells expressing K-RasG12D. Phospho-FACS analysis of these cells revealed markedly increased expression of pAkt when compared to cells expressing K-RasWT or K-RasG12D,Y64G, and similar to pAkt levels in cells expressing K-RasG12D. Preliminary in silico structural analysis of K-RasG12D,Y64G,69RN70 suggests that this novel insertion may restore a critical salt bridge between K-Ras and PI3Kγ.

These data suggest that K-RasG12D oncogenes defective in PI3K signaling are still able to cause dysregulated growth of hematopoietic cells in vitro and in vivo via the acquisition of additional mutations that restore signaling through the PI3K pathway, and strongly support simultaneously targeting multiple downstream effector pathways as a general therapeutic strategy for the substantial fraction of human cancers that contain RAS mutations.

Disclosures:

Bollag: Plexxicon, Inc: Employment, Equity Ownership, Patents & Royalties.