Author Correction: Combination of ERK and autophagy inhibition as a treatment approach for pancreatic cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Low-Dose Vertical Inhibition of the RAF-MEK-ERK Cascade Causes Apoptotic Death of KRAS Mutant Cancers.

We address whether combinations with a pan-RAF inhibitor (RAFi) would be effective in KRAS mutant pancreatic ductal adenocarcinoma (PDAC). Chemical library and CRISPR genetic screens identify combinations causing apoptotic anti-tumor activity. The most potent combination, concurrent inhibition of RAF (RAFi) and ERK (ERKi), is highly synergistic at low doses in cell line, organoid, and rat models of PDAC, whereas each inhibitor alone is only cytostatic. Comprehensive mechanistic signaling studies using reverse phase protein array (RPPA) pathway mapping and RNA sequencing (RNA-seq) show that RAFi/ERKi induced insensitivity to loss of negative feedback and system failures including loss of ERK signaling, FOSL1, and MYC; shutdown of the MYC transcriptome; and induction of mesenchymal-to-epithelial transition. We conclude that low-dose vertical inhibition of the RAF-MEK-ERK cascade is an effective therapeutic strategy for KRAS mutant PDAC.

Combination of ERK and autophagy inhibition as a treatment approach for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth, but the role of KRAS in supporting autophagy has not been established. We show that, to our surprise, suppression of KRAS increased autophagic flux, as did pharmacological inhibition of its effector ERK MAPK. Furthermore, we demonstrate that either KRAS suppression or ERK inhibition decreased both glycolytic and mitochondrial functions. We speculated that ERK inhibition might thus enhance PDAC dependence on autophagy, in part by impairing other KRAS- or ERK-driven metabolic processes. Accordingly, we found that the autophagy inhibitor chloroquine and genetic or pharmacologic inhibition of specific autophagy regulators synergistically enhanced the ability of ERK inhibitors to mediate antitumor activity in KRAS-driven PDAC. We conclude that combinations of pharmacologic inhibitors that concurrently block both ERK MAPK and autophagic processes that are upregulated in response to ERK inhibition may be effective treatments for PDAC.

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.

Rho GTPase Transcriptome Analysis Reveals Oncogenic Roles for Rho GTPase-Activating Proteins in Basal-like Breast Cancers.

The basal-like breast cancer (BLBC) subtype accounts for a disproportionately high percentage of overall breast cancer mortality. The current therapeutic options for BLBC need improvement; hence, elucidating signaling pathways that drive BLBC growth may identify novel targets for the development of effective therapies. Rho GTPases have previously been implicated in promoting tumor cell proliferation and metastasis. These proteins are inactivated by Rho-selective GTPase-activating proteins (RhoGAP), which have generally been presumed to act as tumor suppressors. Surprisingly, RNA-Seq analysis of the Rho GTPase signaling transcriptome revealed high expression of several RhoGAP genes in BLBC tumors, raising the possibility that these genes may be oncogenic. To evaluate this, we examined the roles of two of these RhoGAPs, ArhGAP11A (also known as MP-GAP) and RacGAP1 (also known as MgcRacGAP), in promoting BLBC. Both proteins were highly expressed in human BLBC cell lines, and knockdown of either gene resulted in significant defects in the proliferation of these cells. Knockdown of ArhGAP11A caused CDKN1B/p27-mediated arrest in the G1 phase of the cell cycle, whereas depletion of RacGAP1 inhibited growth through the combined effects of cytokinesis failure, CDKN1A/p21-mediated RB1 inhibition, and the onset of senescence. Random migration was suppressed or enhanced by the knockdown of ArhGAP11A or RacGAP1, respectively. Cell spreading and levels of GTP-bound RhoA were increased upon depletion of either RhoGAP. We have established that, via the suppression of RhoA, ArhGAP11A and RacGAP1 are both critical drivers of BLBC growth, and propose that RhoGAPs can act as oncogenes in cancer. Cancer Res; 76(13); 3826-37. ©2016 AACR.

Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression.

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

CIB1 depletion impairs cell survival and tumor growth in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with generally poor prognosis and no available targeted therapies, highlighting a critical unmet need to identify and characterize novel therapeutic targets. We previously demonstrated that CIB1 is necessary for cancer cell survival and proliferation via regulation of two oncogenic signaling pathways, RAF-MEK-ERK and PI3K-AKT. Because these pathways are often upregulated in TNBC, we hypothesized that CIB1 may play a broader role in TNBC cell survival and tumor growth. Methods utilized include inducible RNAi depletion of CIB1 in vitro and in vivo, immunoblotting, clonogenic assay, flow cytometry, RNA-sequencing, bioinformatics analysis, and Kaplan-Meier survival analysis. CIB1 depletion resulted in significant cell death in 8 of 11 TNBC cell lines tested. Analysis of components related to PI3K-AKT and RAF-MEK-ERK signaling revealed that elevated AKT activation status and low PTEN expression were key predictors of sensitivity to CIB1 depletion. Furthermore, CIB1 knockdown caused dramatic shrinkage of MDA-MB-468 xenograft tumors in vivo. RNA sequence analysis also showed that CIB1 depletion in TNBC cells activates gene programs associated with decreased proliferation and increased cell death. CIB1 expression levels per se did not predict TNBC susceptibility to CIB1 depletion, and CIB1 mRNA expression levels did not associate with TNBC patient survival. Our data are consistent with the emerging concept of non-oncogene addiction, where a large subset of TNBCs depend on CIB1 for cell survival and tumor growth, independent of CIB1 expression levels. Our data establish CIB1 as a novel therapeutic target for TNBC.