Defective K-Ras oncoproteins overcome impaired effector activation to initiate leukemia in vivo

Abstract

Reversing the aberrant biochemical output of oncogenic Ras proteins is one of the great challenges in cancer therapeutics; however, it is uncertain which Ras effectors are required for tumor initiation and maintenance. To address this question, we expressed oncogenic K-Ras(D12) proteins with "second site" amino acid substitutions that impair PI3 kinase/Akt or Raf/MEK/ERK activation in bone marrow cells and transplanted them into recipient mice. In spite of attenuated signaling properties, defective K-Ras oncoproteins initiated aggressive clonal T-lineage acute lymphoblastic leukemia (T-ALL). Murine T-ALLs expressing second site mutant proteins restored full oncogenic Ras activity through diverse mechanisms, which included acquiring novel somatic third site Kras(D12) mutations and silencing PTEN. T-ALL cell lines lacking PTEN had elevated levels of phosphorylated Akt, a gene expression pattern similar to human early T-cell precursor ALL, and were resistant to the potent and selective MEK inhibitor PD0325901. Our data, which demonstrate strong selective pressure to overcome the defective activation of PI3 kinase/Akt and Raf/MEK/ERK, implicate both Ras effector pathways as drivers of aberrant growth in T-ALL and further suggest that leukemia cells will deploy multiple mechanisms to develop resistance to targeted inhibitors in vivo.

Figure 1

Second site Kras^D12 mutant alleles induce hypersensitive myeloid progenitor growth and initiate T-ALL. (A) CFU-GM formation by fetal liver cells expressing WT Kras (●), Kras^D12 (▪), Kras^D12/G37 (▲), or Kras^D12/G64 (♦). Mean ± standard error of the mean (SEM) of 5 independent experiments is shown. Data points marked with an asterisk (*) are significantly different from WT by paired, 1-tailed t test (P < .05). (B) Signaling in transfected COS-7 cells under basal (B), starved (S), and EGF-stimulated (E) conditions. (C) Signaling in transduced S49 cells under starved conditions. These cells were infected with MSCV-GFP-Kras vectors and sorted to equalize Ras expression levels. GFP-K-Ras fusion proteins run at a different molecular weight than endogenous Ras proteins. (D) Morphology of 3T3 cells expressing WT Kras, Kras^D12, Kras^D12/G37, or Kras^D12/G64. Note that Kras^D12 and Kras^D12/G64 induce morphologic changes. (E) Survival of lethally irradiated WT mice transplanted with bone marrow cells transduced with MIG vector (×; n = 8) or MIG vectors expressing WT Kras (●; n = 10), Kras^D12 (▪; n = 9), Kras^D12/G37 (▲; n = 15), or Kras^D12/G64 (♦; n = 14). (F) White blood cell (WBC) counts at death in recipients of bone marrow transduced with MIG vector (n = 8), Kras^D12/G37 (n = 15), or Kras^D12/G64 (n = 14) viruses. Data plotted as mean ± SEM, with an asterisk (*) indicating data points significantly different from WT by unpaired, 1-tailed t test (P < .05). (G) Peripheral blood smear showing blast morphology in a mouse with T-ALL.

Figure 2

Leukemias initiated by Kras^D12/G37 and Kras^D12/G64 expression demonstrate distinct signaling profiles. (A) Immunoblot of T-ALL cell lines under basal (B) and starved (S) conditions. Two control T-ALL cell lines with WT Kras (C) from a retroviral insertional mutagenesis screen were included. Ten cell lines were generated from independent leukemias induced by either Kras^D12/G37 (E1-E4) or Kras^D12/G64 (Y1-Y6). (B) Ras was immunoprecipitated from cell lines E1-E4 and then probed with an antibody that recognizes the D12 substitution. (C) quantitative PCR analysis of Kras expression in T-ALL cell lines compared with WT thymus.

Figure 3

Somatic third site Kras mutations in T-ALL. In cell lines E1 (A) and E3 (B), the single amino acid substitution T50I was present at ∼50% frequency based on relative abundance of sequence reads. It was detected at a similar frequency in the spleens of the secondary recipients used to generate these cell lines and in a single primary recipient. (C) The sequence GAGACC was inserted between amino acids 69 and 70 of Kras in T-ALL cell line Y4. This mutation is present in ∼50% of Kras transcripts in cell line Y4, at a lower frequency in the spleen of the secondary recipient used to generate this cell line, and is not seen in the primary leukemia.

Figure 4

Acquired third site mutations restore oncogenic activity to Kras^D12/G37 and Kras^D12/G64. (A) CFU-GM formation of fetal liver cells expressing WT Kras (●), Kras^D12 (▪), Kras^D12/G37/I50 (Δ), or Kras^{D12/G64/69RN70} (♢). Data show the mean of 3 independent experiments. (B) Representative CFU-GM morphology from fetal liver cells expressing Kras mutant alleles grown in 0.1 ng/mL GM-CSF. (C) Ras expression in GFP+, Mac1+ fetal liver cells infected with MSCV viruses encoding different Kras alleles. (D) Levels of pERK, pAkt, and pS6 in GFP+, Mac1+ fetal liver cells infected with MSCV viruses encoding different Kras alleles, as determined by flow cytometry using phospho-specific antibodies. Phospho-protein levels in cells expressing WT K-Ras were set at 1 in each experiment. Data shown are derived from 6 independent experiments.

Figure 5

PTEN loss confers resistance to inhibition of MEK but not PI3K or Akt. IC₅₀s and mean growth curves of T-ALL cell lines with and without PTEN expression in varying doses of (A) the PI3K inhibitor GDC-0941, (B) the Akt inhibitor MK2206, and (C) the MEK inhibitor PD0325901. (D) PD0325901 induces apoptosis in PTEN-positive, but not PTEN-negative, cell lines. Curves indicate mean growth of 4 PTEN-positive cell lines (E1, E2, E3, and Y4) and 6 PTEN-negative cell lines (E4, Y1, Y2, Y3, Y5, and Y6) ± SEM. ***P < .001, *P < .05.

Figure 6

PI3K-activated T-ALL cell lines and human ETP ALLs have similar gene expression profiles. (A) Principal component analysis of the gene expression profiling data of all 10 mouse T-ALL cell lines using 200 representative genes selected by k-means algorithm, showing cases clustered according to PI3K activation status (red, activated; blue, not activated). (B) Gene set enrichment analysis demonstrates significant enrichment of the top 100 mouse PI3K up-regulated genes in ETP ALL (P = .057; false discovery rate, 0.224). (C) Heat map of the leading-edge mouse PI3K up-regulated genes in gene set enrichment analysis, showing overexpression of these genes in ETP ALL.