SUMMARY Kinase inhibitors have limited success in cancer treatment because tumors circumvent their action. Using a quantitative proteomics approach, we assessed kinome activity in response to MEK inhibition in triple negative breast cancer (TNBC) cells and genetically engineered mice (GEMMs). MEK inhibition caused acute ERK activity loss, resulting in rapid c-Myc degradation that induced expression and activation of several receptor tyrosine kinases (RTKs). RNAi knockdown of ERK or c-Myc mimicked RTK induction by MEK inhibitors, whereas prevention of proteasomal c-Myc degradation blocked kinome reprogramming. MEK inhibitor-induced RTK stimulation overcame MEK2 but not MEK1 inhibition, reactivating ERK and producing drug resistance. The C3Tag GEMM for TNBC similarly induced RTKs in response to MEK inhibition. The inhibitor-induced RTK profile suggested a kinase inhibitor combination therapy that produced GEMM tumor apoptosis and regression where single agents were ineffective. This approach defines mechanisms of drug resistance, allowing rational design of combination therapies for cancer.

SUMMARY Therapeutics such as lapatinib that target ERBB2 often provide initial clinical benefit but resistance frequently develops. Adaptive responses leading to lapatinib resistance involve reprogramming of the kinome through reactivation of ERBB2/ERBB3 signaling and transcriptional upregulation and activation of multiple tyrosine kinases. The heterogeneity of induced kinases prevents their targeting by a single kinase inhibitor, underscoring the challenge of predicting effective kinase inhibitor combination therapies. We hypothesized that to make the tumor response to single kinase inhibitors durable, the adaptive kinome response itself must be inhibited. Genetic and chemical inhibition of BET bromodomain chromatin readers suppresses transcription of many lapatinib-induced kinases involved in resistance including ERBB3, IGF1R, DDR1, MET, and FGFRs, preventing downstream SRC/FAK signaling and AKT reactivation. Combining inhibitors of kinases and chromatin readers prevents kinome adaptation by blocking transcription, generating a durable response to lapatinib and overcoming the dilemma of heterogeneity in the adaptive response.

Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated thin-walled blood vessels that predispose individuals to seizures and stroke. Familial CCM is caused by mutations in KRIT1 (CCM1) or in malcavernin (CCM2), the murine ortholog of which was concurrently characterized as osmosensing scaffold for MEKK3 (OSM). The roles of the CCM proteins in the pathogenesis of the disorder remain largely unknown. Here, we use co-immunoprecipitation, fluorescence resonance energy transfer and subcellular localization strategies to show that the CCM1 gene product, KRIT1, interacts with the CCM2 gene product, malcavernin/OSM. Analogous to the established interactions of CCM1 and beta1 integrin with ICAP1, the CCM1/CCM2 association is dependent upon the phosphotyrosine binding (PTB) domain of CCM2. A familial CCM2 missense mutation abrogates the CCM1/CCM2 interaction, suggesting that loss of this interaction may be critical in CCM pathogenesis. CCM2 and ICAP1 bound to CCM1 via their respective PTB domains differentially influence the subcellular localization of CCM1. Furthermore, we expand upon the established involvement of CCM2 in the p38 mitogen-activated protein kinase signaling module by demonstrating that CCM1 associates with CCM2 and MEKK3 in a ternary complex. These data indicate that the genetic heterogeneity observed in familial CCM may reflect mutation of different molecular members of a coordinated signaling complex.

Cerebral cavernous malformations (CCMs) are congenital vascular anomalies of the central nervous system that can result in hemorrhagic stroke, seizures, recurrent headaches, and focal neurologic deficits. Mutations in the gene KRIT1 are responsible for type 1 CCM (CCM1). We report that a novel gene, MGC4607, exhibits eight different mutations in nine families with type 2 CCM (CCM2). MGC4607, similar to the KRIT1 binding partner ICAP1alpha, encodes a protein with a phosphotyrosine-binding domain. This protein may be part of the complex pathway of integrin signaling that, when perturbed, causes abnormal vascular morphogenesis in the brain, leading to CCM formation.

Targeting the dysregulated BRaf-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRaf and MEK, resistance develops often involving non-genomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in triple negative breast cancer (TNBC) patients induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTKs) comparing tumor samples before and after one week of treatment. In preclinical models MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation and p300 that drives transcriptional adaptation. Inhibition of P-TEFb associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts and syngeneic mouse TNBC models. Pharmacological targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.

Mutations in KRIT1, a protein initially identified based on a yeast two-hybrid interaction with the RAS-family GTPase RAP1A, are responsible for the development of the inherited vascular disorder cerebral cavernous malformations (CCM1). As the function of the KRIT1 protein and its role in CCM pathogenesis remain unknown, we performed yeast two-hybrid screens to identify additional protein binding partners. A fragment containing the N-terminal 272 amino acid residues of KRIT1, a region lacking similarity to any known protein upon database searches, was used as bait. From parallel screens of human fetal brain and HeLa cDNA libraries, we obtained multiple independent isolates of human integrin cytoplasmic domain-associated protein-1 (ICAP-1) as interacting clones. The interaction of KRIT1 and ICAP-1 was confirmed by GST-KRIT1 trapping of endogenous ICAP-1 from 293T cells. The alpha isoform of ICAP-1 is a 200 amino acid serine/threonine-rich phosphoprotein which binds the cytoplasmic tail of beta1 integrins. We show that mutagenesis of the N-terminal KRIT1 NPXY amino acid sequence, a motif critical for ICAP-1 binding to beta1 integrin molecules, completely abrogates the KRIT1/ICAP-1 interaction. The interaction between ICAP-1 and KRIT1, and the presence of a FERM domain in the latter, suggest that KRIT1 might be involved in the bidirectional signaling between integrin molecules and the cytoskeleton. Furthermore, these data suggest that KRIT1 might affect cell adhesion processes via integrin signaling in CCM1 pathogenesis.

Advances in our understanding of fundamental biological processes can be made by the analysis of defects manifested in inherited diseases. The genes responsible for these genetic syndromes often encode proteins that act at critical points of the pathways that control biological processes such as cell proliferation, cell-cell communication, cellular differentiation, and cell death. This approach has lead to the discovery of novel gene products and/or biochemical pathways involved in disease, genes that in turn play a fundamental role in normal biological processes. This forward genetic approach, focusing on Mendelian disorders of vascular anomalies, has been particularly fruitful for the study of genetic regulation of angiogenesis. This review summarizes the ongoing saga of two genetic syndromes involving disruption of normal vascular morphogenesis. Each inherited disorder involves the focal development of a distinct vascular anomaly. In hereditary hemorrhagic telangiectasia (HHT), the hallmark vascular lesion is termed an arteriovenous malformation, which involves the direct communication of an artery with a vein (arteriovenous shunt), without an intervening capillary bed. For cerebral cavernous malformations (CCM), the lesions are grossly-dilated, closely-packed, capillary-like sinusoidal chambers. The autosomal dominant mode of inheritance of each of these distinct syndromes suggested that the underlying genes might regulate critical aspects of vascular morphogenesis. Emerging but intriguing tales are being told by the genes (and their protein products) mutated in these disorders.

The central role of the BRAF-MEK-ERK pathway in controlling cell fate has made this pathway a primary target for deregulated activation in cancer. BRaf is activated by Ras proteins allowing Ras oncogenes to constitutively activate the pathway. Activating BRaf mutations are also frequent in several cancers, being the most common oncogenic mutation in thyroid carcinoma and melanoma. There are currently two inhibitors, vemurafenib and dabrafenib, approved for treatment of malignant melanoma having activating BRaf mutations. Concurrent administration of BRAF inhibitor and MEK inhibitor (trametinib) is significantly more active in patients with BRAF mutant melanoma than either single agent alone, but progression to resistance ultimately occurs by different mechanisms that increase the activation of ERK. Such adaptive changes in tumor cell signaling networks allows bypass of targeted oncoprotein inhibition. This is true with targeted inhibitors for BRaf and MEK as well as specific inhibitors for AKT, mTOR and many receptor tyrosine kinases such as EGFR and HER2. It is this adaptive response to targeted kinase inhibitors that contributes to the failure of single agent kinase inhibitors to have durable responses. This failure is seen in virtually all cancers treated with single agent kinase inhibitors, most of which are not as dependent on a single signaling pathway such as BRaf-MEK-ERK in melanoma. Thus, understanding the breadth of adaptive reprogramming responses to specific targeted kinase inhibition will be critical to develop appropriate combination therapies for durable clinical responses.