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. 2011 Feb 24;117(8):2433-40.
doi: 10.1182/blood-2010-09-305128. Epub 2011 Jan 5.

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Michael K Kiessling  1 , Patrick A Oberholzer, Chandrani Mondal, Maria B Karpova, Marie C Zipser, William M Lin, Michael Girardi, Laura E Macconaill, Sarah M Kehoe, Charlie Hatton, Lars E French, Levi A Garraway, Gernot Polier, Dorothee Süss, Claus-Detlev Klemke, Peter H Krammer, Karsten Gülow, Reinhard Dummer
Affiliations

High-throughput mutation profiling of CTCL samples reveals KRAS and NRAS mutations sensitizing tumors toward inhibition of the RAS/RAF/MEK signaling cascade

Michael K Kiessling et al. Blood. .

Abstract

Cutaneous T-cell lymphomas (CTCLs) are malignancies of skin-homing lymphoid cells, which have so far not been investigated thoroughly for common oncogenic mutations. We screened 90 biopsy specimens from CTCL patients (41 mycosis fungoides, 36 Sézary syndrome, and 13 non-mycosis fungoides/Sézary syndrome CTCL) for somatic mutations using OncoMap technology. We detected oncogenic mutations for the RAS pathway in 4 of 90 samples. One mycosis fungoides and one pleomorphic CTCL harbored a KRAS(G13D) mutation; one Sézary syndrome and one CD30(+) CTCL harbored a NRAS(Q61K) amino acid change. All mutations were found in stage IV patients (4 of 42) who showed significantly decreased overall survival compared with stage IV patients without mutations (P = .04). In addition, we detected a NRAS(Q61K) mutation in the CTCL cell line Hut78. Knockdown of NRAS by siRNA induced apoptosis in mutant Hut78 cells but not in CTCL cell lines lacking RAS mutations. The NRAS(Q61K) mutation sensitized Hut78 cells toward growth inhibition by the MEK inhibitors U0126, AZD6244, and PD0325901. Furthermore, we found that MEK inhibitors exclusively induce apoptosis in Hut78 cells. Taken together, we conclude that RAS mutations are rare events at a late stage of CTCL, and our preclinical results suggest that such late-stage patients profit from MEK inhibitors.

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Figures

Figure 1
Figure 1
CTCL stage IV patients harboring a RAS mutation show reduced life expectancy. Kaplan-Meier curve: Comparison of survival of mutant (all stage IV), nonmutant stage IV patients (P = .041), and nonmutant stage I to III patients (P = .001). P values were determined by Student t test (2-tailed) comparing individual group versus the mutant patient samples group. Similarly, the 2 nonmutant groups, when considered together, showed significance for prolonged survival compared with the mutant patient samples group (P = .017, 2-tailed Student t test). Considered are time span from first diagnosis to date of death or last visit, when applicable, respectively.
Figure 2
Figure 2
CTCL cell line Hut78 harbors Q61K mutation for NRAS. (A) RNA was isolated and reverse transcribed to cDNA for all 4 cell lines. Next, PCR was performed covering the open reading frame of KRAS, NRAS, HRAS, and BRAF. PCR products were purified and sent for sequencing. Results were evaluated by Genetic Analysis Technology Consortium sequence viewer. Arrow indicates cytosine-to-adenosine conversion. (B) Table of sequencing results for all 4 CTCL cell lines. A heterozygous Q61K mutation was detected for the Hut78 cell line. A homozygous silent H25H mutation was found in the SeAx cell line. (C) All 4 cell lines were kept under equal conditions, lysed, and and lysates subjected to Western blot. Phosphorylation of ERK was assayed by anti-phospho-specific ERK antibodies. Equal expression of ERK was verified by anti-ERK2 antibodies, and equal loading was verified by anti–tubulin antibodies. Quantification of phosphorylated ERK to ERK2 was done with ImageJ 1.43 Quantification software. (D) Same as panel C, but anti-phospho-specific MEK antibodies and anti-MEK antibodies were used to assess the phosphorylation status of MEK and MEK expression, respectively. Equal loading was verified by antitubulin antibodies.
Figure 3
Figure 3
NRASQ61K is required for survival of the CTCL cell line Hut78. (A) siRNA against NRAS (siNRAS1 and siNRAS2) were transfected in SeAx, Hut78, MyLa, and HH cell lines by the Amaxa kit and protocol. At 72 hours after transfection, cells were lysed and lysates subjected to Western blot. Efficiency of knockdown was measured by anti-NRAS antibodies. Levels of phosphorylation of MEK and ERK were assessed by anti-phospho-specific MEK and anti-phospho-specific ERK in SeAx and Hut78 cells. Equal loading was verified by antitubulin antibodies. (B) Apoptosis was measured starting 3 days after siRNA transfection for 24, 48, and 72 hours. Specific apoptosis was calculated as described in “Cell death assays.” Data are representative of 3 independent experiments.
Figure 4
Figure 4
NRASQ61K sensitizes for treatment with MEK inhibitors. (A) Hut78 cells were left untreated or treated with 1μM AZD6244, 1μM PD0325901, and 1μM U0126 for 4 hours. Then, cells were lysed, and the basal phosphorylation level of ERK and MEK was assessed by Western blot with specific anti-phospho-ERK and with specific anti-phospho-MEK antibodies. Equal loading was verified by antitubulin antibodies. (B-D) CTCL cell lines were incubated with indicated concentrations of MEK inhibitors U0126 (B), AZD6244 (C), and PD0325901 (D) for 72 hours. Cell growth was measured by Cell Titer Glo according to the manufacturer's instructions. The 50% inhibitory concentration values, at which 50% of the cell growth inhibition compared with dimethyl sulfoxide was observed, were calculated by GraphPad Prism Version 5 software. Data are representative for 3 independent experiments.
Figure 5
Figure 5
NRASQ61K sensitizes for apoptosis by MEK inhibitors. (A-C) All 4 CTCL cell lines were incubated with indicated concentrations of MEK inhibitors U0126 (A), AZD6244 (B), and PD0325901 (C) for 48 hours (left) and 72 hours (right). Then, apoptosis was determined, and specific apoptosis was calculated as described in “Cell death assays.” Data are representative at least 3 independent experiments.
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