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A novel signalling screen demonstrates that CALR mutations activate essential MAPK signalling and facilitate megakaryocyte differentiation

View Article: PubMed Central - PubMed

ABSTRACT

Most myeloproliferative neoplasm (MPN) patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells, we developed a novel strategy (KISMET) that utilizes the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional small-interfering RNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the mitogen activated protein kinase (MAPK) pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by myeloproliferative leukemia protein (MPL)-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34 cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.

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A screen of cancer cell lines with known kinase addictions proves high reliability and robustness of KISMET. (a) List of top 10 kinases as ranked by the algorithm for the JAK2-dependent cell line HEL and the BCR-ABL1-dependent cell line Ku812. (b) Thirty-six cell lines with known kinase addictions (based on inhibitor studies and/or RNAi mediated knock down of the respective kinase) were screened with KISMET. HL-60 and EOL-1 have been included twice since they have been shown to be addicted to two kinases. The graph shows the rank of the essential kinase for each cell line, as calculated by the algorithm. (c) Each column is composed of 44 dots, each of which represents the rank of the kinase indicated below for one of 44 screened cell lines. Orange or red coloured dots represent the kinase ranks of cell lines known to be addicted to the indicated kinase; white and grey dots depict kinase ranks of cell lines not known to be addicted to the indicated kinase. (d) The CALR-mutant cell line MARIMO has been screened with KISMET. Shown are the kinase ranks for the MAPK members MEK1, MEK2, ERK1, ERK2 and RAF1 in MARIMO cells as well as 12 other human myeloid cell lines. (e, f) MARIMO, HEL (JAK2V617F+) and K562 (BCR-ABL1+) cells have been used to perform 10-point dose–response assays with the MEK1/2 and ERK1/2 inhibitor AZD 6244 (e) or the MEK1/2 inhibitor PD0325901 (f). The tables below show the IC50 values for all three cell lines upon 72 h of treatment. (g) The haematopoietic human cell lines UKE-1 and SET-2 (both JAK2V617F+ MPN), HL-60 (NRAS Q61L+ AML) as well as MARIMO, HEL and K562 cells have been analysed by western blot for their MEK1/2 and ERK1/2 activation.
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fig2: A screen of cancer cell lines with known kinase addictions proves high reliability and robustness of KISMET. (a) List of top 10 kinases as ranked by the algorithm for the JAK2-dependent cell line HEL and the BCR-ABL1-dependent cell line Ku812. (b) Thirty-six cell lines with known kinase addictions (based on inhibitor studies and/or RNAi mediated knock down of the respective kinase) were screened with KISMET. HL-60 and EOL-1 have been included twice since they have been shown to be addicted to two kinases. The graph shows the rank of the essential kinase for each cell line, as calculated by the algorithm. (c) Each column is composed of 44 dots, each of which represents the rank of the kinase indicated below for one of 44 screened cell lines. Orange or red coloured dots represent the kinase ranks of cell lines known to be addicted to the indicated kinase; white and grey dots depict kinase ranks of cell lines not known to be addicted to the indicated kinase. (d) The CALR-mutant cell line MARIMO has been screened with KISMET. Shown are the kinase ranks for the MAPK members MEK1, MEK2, ERK1, ERK2 and RAF1 in MARIMO cells as well as 12 other human myeloid cell lines. (e, f) MARIMO, HEL (JAK2V617F+) and K562 (BCR-ABL1+) cells have been used to perform 10-point dose–response assays with the MEK1/2 and ERK1/2 inhibitor AZD 6244 (e) or the MEK1/2 inhibitor PD0325901 (f). The tables below show the IC50 values for all three cell lines upon 72 h of treatment. (g) The haematopoietic human cell lines UKE-1 and SET-2 (both JAK2V617F+ MPN), HL-60 (NRAS Q61L+ AML) as well as MARIMO, HEL and K562 cells have been analysed by western blot for their MEK1/2 and ERK1/2 activation.

Mentions: To validate KISMET, we initially focused on six cell lines known to be dependent on either the JAK2V617F mutation (UKE-1, HEL and SET-2)31 or the BCR-ABL1 fusion (Ku812, K562 and LAMA84).32 For HEL and Ku812 the top 10 kinases ranked by their kinase score are shown in Figure 2a. KISMET ranked JAK2 at position 1, 2 and 2 in the three JAK2V617F mutant cell lines and ABL1 at position 1 in all three BCR-ABL1-positive cell lines (Supplementary Figures S2A and B). The complete kinase ranking of all cell lines is shown in Supplementary Data Set S2.


A novel signalling screen demonstrates that CALR mutations activate essential MAPK signalling and facilitate megakaryocyte differentiation
A screen of cancer cell lines with known kinase addictions proves high reliability and robustness of KISMET. (a) List of top 10 kinases as ranked by the algorithm for the JAK2-dependent cell line HEL and the BCR-ABL1-dependent cell line Ku812. (b) Thirty-six cell lines with known kinase addictions (based on inhibitor studies and/or RNAi mediated knock down of the respective kinase) were screened with KISMET. HL-60 and EOL-1 have been included twice since they have been shown to be addicted to two kinases. The graph shows the rank of the essential kinase for each cell line, as calculated by the algorithm. (c) Each column is composed of 44 dots, each of which represents the rank of the kinase indicated below for one of 44 screened cell lines. Orange or red coloured dots represent the kinase ranks of cell lines known to be addicted to the indicated kinase; white and grey dots depict kinase ranks of cell lines not known to be addicted to the indicated kinase. (d) The CALR-mutant cell line MARIMO has been screened with KISMET. Shown are the kinase ranks for the MAPK members MEK1, MEK2, ERK1, ERK2 and RAF1 in MARIMO cells as well as 12 other human myeloid cell lines. (e, f) MARIMO, HEL (JAK2V617F+) and K562 (BCR-ABL1+) cells have been used to perform 10-point dose–response assays with the MEK1/2 and ERK1/2 inhibitor AZD 6244 (e) or the MEK1/2 inhibitor PD0325901 (f). The tables below show the IC50 values for all three cell lines upon 72 h of treatment. (g) The haematopoietic human cell lines UKE-1 and SET-2 (both JAK2V617F+ MPN), HL-60 (NRAS Q61L+ AML) as well as MARIMO, HEL and K562 cells have been analysed by western blot for their MEK1/2 and ERK1/2 activation.
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fig2: A screen of cancer cell lines with known kinase addictions proves high reliability and robustness of KISMET. (a) List of top 10 kinases as ranked by the algorithm for the JAK2-dependent cell line HEL and the BCR-ABL1-dependent cell line Ku812. (b) Thirty-six cell lines with known kinase addictions (based on inhibitor studies and/or RNAi mediated knock down of the respective kinase) were screened with KISMET. HL-60 and EOL-1 have been included twice since they have been shown to be addicted to two kinases. The graph shows the rank of the essential kinase for each cell line, as calculated by the algorithm. (c) Each column is composed of 44 dots, each of which represents the rank of the kinase indicated below for one of 44 screened cell lines. Orange or red coloured dots represent the kinase ranks of cell lines known to be addicted to the indicated kinase; white and grey dots depict kinase ranks of cell lines not known to be addicted to the indicated kinase. (d) The CALR-mutant cell line MARIMO has been screened with KISMET. Shown are the kinase ranks for the MAPK members MEK1, MEK2, ERK1, ERK2 and RAF1 in MARIMO cells as well as 12 other human myeloid cell lines. (e, f) MARIMO, HEL (JAK2V617F+) and K562 (BCR-ABL1+) cells have been used to perform 10-point dose–response assays with the MEK1/2 and ERK1/2 inhibitor AZD 6244 (e) or the MEK1/2 inhibitor PD0325901 (f). The tables below show the IC50 values for all three cell lines upon 72 h of treatment. (g) The haematopoietic human cell lines UKE-1 and SET-2 (both JAK2V617F+ MPN), HL-60 (NRAS Q61L+ AML) as well as MARIMO, HEL and K562 cells have been analysed by western blot for their MEK1/2 and ERK1/2 activation.
Mentions: To validate KISMET, we initially focused on six cell lines known to be dependent on either the JAK2V617F mutation (UKE-1, HEL and SET-2)31 or the BCR-ABL1 fusion (Ku812, K562 and LAMA84).32 For HEL and Ku812 the top 10 kinases ranked by their kinase score are shown in Figure 2a. KISMET ranked JAK2 at position 1, 2 and 2 in the three JAK2V617F mutant cell lines and ABL1 at position 1 in all three BCR-ABL1-positive cell lines (Supplementary Figures S2A and B). The complete kinase ranking of all cell lines is shown in Supplementary Data Set S2.

View Article: PubMed Central - PubMed

ABSTRACT

Most myeloproliferative neoplasm (MPN) patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells, we developed a novel strategy (KISMET) that utilizes the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional small-interfering RNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the mitogen activated protein kinase (MAPK) pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by myeloproliferative leukemia protein (MPL)-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34 cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.

No MeSH data available.


Related in: MedlinePlus