A CRISPR screen identifies MAPK7 as a target for combination with MEK inhibition in <i>KRAS</i> mutant NSCLC

<div><p>Mutant <i>KRAS</i> represents one of the most frequently observed oncogenes in NSCLC, yet no therapies are approved for tumors that express activated KRAS variants. While there is strong rationale for the use of MEK inhibitors to treat tumors with activated RAS/MAPK signaling, these have proven ineffective clinically. We therefore implemented a CRISPR screening approach to identify novel agents to sensitize <i>KRAS</i> mutant NSCLC cells to MEK inhibitor treatment. This approach identified multiple components of the canonical RAS/MAPK pathway consistent with previous studies. In addition, we identified <i>MAPK7</i> as a novel, strong hit and validated this finding using multiple orthogonal approaches including knockdown and pharmacological inhibition. We show that MAPK7 inhibition attenuates the re-activation of MAPK signaling occurring following long-term MEK inhibition, thereby illustrating that MAPK7 mediates pathway reactivation in the face of MEK inhibition. Finally, genetic knockdown of MAPK7 combined with the MEK inhibitor cobimetinib in a mutant <i>KRAS</i> NSCLC xenograft model to mediate improved tumor growth inhibition. These data highlight that MAPK7 represents a promising target for combination treatment with MEK inhibition in <i>KRAS</i> mutant NSCLC.</p></div

Inhibition of screen hits leads to reduced pathway rebound and is enhanced with longer term inhibition.

<p><b>(A)</b> Dose response curves at 72 hrs in the presence of inhibitors to RAF, PAK1/2, p38 and MAPK7 +/- 0.25 μM MEK inhibitor. <b>(B)</b> 10 day clonogenic assay with dose response of RAF, PAK1/2, p38 and MAPK7 inhibitors +/- 0.25 μM MEK inhibitor. (<b>C)</b> Western blots showing changes in pERK1/2 levels with indicated inhibitors and time points indicated.</p

Model for the relationship between MEK1/2 inhibition and MAPK7 inhibition.

<p>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.</p

Loss of MAPK7 in combination with MEK inhibition leads to reduced viability in vitro and in vivo.

<p><b>(A</b>) 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 <i>MAPK7</i>. Right: Western blot showing the level of MAPK7 protein loss observed with the labeled gRNA sequence at day 10 for each cell line. <b>(B)</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 <i>MAPK7</i>. Right: Western blot showing the level of MAPK7 protein loss observed with the indicated shRNA sequence at day 10 for both cell lines. <b>(C)</b> 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. <b>(D)</b> 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.</p

A small set of genes lead to changes in MAPK pathway inhibitor sensitivity.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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.</p

Inhibition of MAPK7 in combination with MEK inhibition leads to reduced MAPK pathway rebound and reduced viability.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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. <b>(D)</b> 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.</p

Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody–Drug Conjugates

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) <i>in vitro</i> in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between <i>in vitro</i> GSH stability and <i>in vitro</i> cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody–drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells <i>in vitro</i> in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 <i>in vivo</i> efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties <i>in vivo</i> relative to the parent ADC, which did not contain the prodrug