Fig 1.
A small set of genes lead to changes in MAPK pathway inhibitor sensitivity.
(A) Waterfall plot showing the median log fold-change (LFC) in gRNAs targeting each of the ~2200 genes in the library in response to cobimetinib. NTC gRNAs indicated in bold, enlarged are the genes with >1 LFC either in abundance or depletion. (B) Examples of the performance of individual gRNAs for genes both enriched and depleted from the pool. Individual gRNAs in the presence of DMSO in thin solid lines, dashed lines in the presence of cobimetinib, thick lines represent the median values. (C) Using a >0.5 Median LFC cutoff this Venn-diagram summarizes the small set of genes whose abundance change (depletion in red, enrichment in blue) occurred in both inhibitor arms of the screen. A much larger set of genes showed changes only in the MEK inhibitor arm.
Fig 2.
Validation of genes regulating MAPK inhibitor sensitivity in multiple cell lines.
(A) Four cell lines were subjected to a sub-library pooled screen (mini-pool). The performance of gRNAs in the MOR cell line is compared to activity observed in the original screen. The additional cell lines were then compared to the “mini-pool” performance of the MOR cell line. (B) Bar plot showing the combination performance of each of the guides in the sub-library in an arrayed format, at passage 3 in A549 cells. A heat map shows the average combination score across the four guides targeting each gene, across the four passages of the array screen. (C) Top: Bar plots showing sensitivity changes in the presence of MEKi with siRNA targeting several hits from the screen. Bottom: combination scores for the siRNA assay.
Fig 3.
Inhibition of screen hits leads to reduced pathway rebound and is enhanced with longer term inhibition.
(A) Dose response curves at 72 hrs in the presence of inhibitors to RAF, PAK1/2, p38 and MAPK7 +/- 0.25 μM MEK inhibitor. (B) 10 day clonogenic assay with dose response of RAF, PAK1/2, p38 and MAPK7 inhibitors +/- 0.25 μM MEK inhibitor. (C) Western blots showing changes in pERK1/2 levels with indicated inhibitors and time points indicated.
Fig 4.
Inhibition of MAPK7 in combination with MEK inhibition leads to reduced MAPK pathway rebound and reduced viability.
(A) Left: Dose response curves at 72 hrs for MAPK7 inhibitor +/- MEK inhibitor for NCI-H441 and NCI-H2122 cells. Right: 10 day clonogenic assay for the same combinations as the 72 hrs assay. (B) Western blot showing MAPK7 phosphorylation (band shift) and ERK1/2 phosphorylation in response to MEK inhibitor (0.25 μM), MAPK7 inhibitor (1 μM) and the combination at four time-points in A549, NCI-H2122, NCI-H441 and MOR cell lines. (C) Western blot showing phosphorylation changes in response to EGF addition (O/N starve, 10 min stimulation), MEK inhibitor (0.25 μM, 24 hrs), MAPK7 inhibitor (1 μM, 24 hrs) and combination, in the A549 cell line. (D) Western blot showing MAPK7 phosphorylation (band shift) in response to MEK inhibitor (0.25 μM), EGFR inhibitor (1 μM) and combination at four time points in the A549 cell line.
Fig 5.
Loss of MAPK7 in combination with MEK inhibition leads to reduced viability in vitro and in vivo.
(A) Left: Long-term growth assay showing the effect on cell proliferation at day 10 in NCI-H2122, MOR, A549, and NCI-H441 with MEK inhibitor alone and in combination with a control gRNA sequence and two sequences targeting MAPK7. Right: Western blot showing the level of MAPK7 protein loss observed with the labeled gRNA sequence at day 10 for each cell line. (B) Left: Long-term growth assay showing the effect on cell proliferation at day 10 in NCI-H2122 and NCI-H441 with MEK inhibitor alone and in combination with inducible shRNA mediated knockdown of MAPK7. Right: Western blot showing the level of MAPK7 protein loss observed with the indicated shRNA sequence at day 10 for both cell lines. (C) Plot showing NCI-H2122 shMAPK7 xenograft tumor volumes for tumors treated with vehicle or treated with MEK inhibitor, and in the presence or absence of doxycycline (Dox) induction of shRNA mediated knockdown. Tumor volumes are summarized using a mixed linear effects model. (D) Western blot showing levels and phosphorylation of MAPK7 and phosphorylation of ERK1/2 in four tumor samples from each arm at the end of the study. Right: Bar plot showing quantitation of ßactin normalized levels of pERK1/2 from the tumors shown in the western blot in the center.
Fig 6.
Model for the relationship between MEK1/2 inhibition and MAPK7 inhibition.
In untreated proliferating cells all signaling components show basal activity and active ERK1/2 relay negative feedback signals upstream of Raf kinases. Following MEK inhibitor treatment, ERK activity is reduced, decreasing cell proliferation, but relieving negative feedback to RTKs/Raf and MAPK7. Eventually this increased MAPK7 activity can contribute to ERK1/2 reactivation even in the presence of MEK inhibitors. MAPK7 knockout/knockdown or inhibition prevents the delayed reactivation of ERK1/2 in the presence of MEK inhibitors thereby causing a more pronounced inhibition of cell proliferation, especially in long term assays.