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Mutations in MEK1/2 have been described as a resistance mechanism to BRAF/MEK inhibitor treatment. We report the discovery of a novel ATP-competitive MEK1/2 inhibitor with efficacy in wildtype (WT) and mutant MEK12 models. Starting from a HTS hit, we obtained selective, cellularly active compounds that showed equipotent inhibition of WT MEK1/2 and a panel of MEK1/2 mutant cell lines. Using a structure-based approach, the optimization addressed the liabilities by systematic analysis of molecular matched pairs (MMPs) and ligand conformation. Addition of only three heavy atoms to early tool compound

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CD3-bispecific antibodies represent an important therapeutic strategy in oncology. These molecules work by redirecting cytotoxic T cells to antigen-bearing tumor cells. Although CD3-bispecific antibodies have been developed for several clinical indications, cases of cancer-derived resistance are an emerging limitation to the more generalized application of these molecules. Here, we devised whole-genome CRISPR screens to identify cancer resistance mechanisms to CD3-bispecific antibodies across multiple targets and cancer types. By validating the screen hits, we found that deficiency in IFNγ signaling has a prominent role in cancer resistance. IFNγ functioned by stimulating the expression of T-cell killing–related molecules in a cell type–specific manner. By assessing resistance to the clinical CD3-bispecific antibody flotetuzumab, we identified core fucosylation as a critical pathway to regulate flotetuzumab binding to the CD123 antigen. Disruption of this pathway resulted in significant resistance to flotetuzumab treatment. Proper fucosylation of CD123 was required for its normal biological functions. In order to treat the resistance associated with fucosylation loss, flotetuzumab in combination with an alternative targeting CD3-bispecific antibody demonstrated superior efficacy. Together, our study reveals multiple mechanisms that can be targeted to enhance the clinical potential of current and future T-cell–engaging CD3-bispecific antibody therapies.

PDF file - 416KB, Supplementary methods, figures and tables. Fig. S1. Gene expression profiling of patient-matched brain and extracranial metastases. Fig. S2. PTEN gene expression in BM_03 (homozygous copy loss) and EM_03 (one copy loss). Gene expression was measured by microarray. Fig. S3. Quantitative RT-PCR measurement of SGK3, SGSM2, and ELOVL2 expression in five pairs of brain metastases (BM) and extracranial metastases (EM). Fig. S4. Log2 expression levels of RB_pS807_S811 in seven pairs of matched metastases. Fig. S5 Examples of PTEN-absent tumors. PTEN IHC of BM_03 and EM_03 were photographed at 00X magnification. Fig. S6 GSK3/_pS21/S9 IHC staining in 26 pairs of matched brain metastases (BM) and extracranial metastases (EM). Fig. S7. Results of RB_pS807_S811 IHC in 25 pairs of matched brain metastases (BM) and extracranial metastases (EM). Fig. S8. Kaplan-Meier survival analysis of nude mice with BRAF-mutant melanoma brain metastases treated with BKM120 and/or LGX818. Fig. S9. AKT_pS473 level in subcutaneously (sc) and intracranially (ic) implanted melanomas. Table S1. List of clinical samples analyzed. Table S2. Clinical features and genetic aberrations of matched samples analyzed by high-throughput techniques. Table S3. Hotspot mutations analyzed by Sequenom. Table S4. Antibodies used in RPPA analysis. Table S5. Genes with copy number differences (P

Purpose: An improved understanding of the molecular pathogenesis of brain metastases, one of the most common and devastating complications of advanced melanoma, may identify and prioritize rational therapeutic approaches for this disease. In particular, the identification of molecular differences between brain and extracranial metastases would support the need for the development of organ-specific therapeutic approaches.Experimental Design: Hotspot mutations, copy number variations (CNV), global mRNA expression patterns, and quantitative analysis of protein expression and activation by reverse-phase protein array (RPPA) analysis were evaluated in pairs of melanoma brain metastases and extracranial metastases from patients who had undergone surgical resection for both types of tumors.Results: The status of 154 previously reported hotspot mutations, including driver mutations in BRAF and NRAS, were concordant in all evaluable patient-matched pairs of tumors. Overall patterns of CNV, mRNA expression, and protein expression were largely similar between the paired samples for individual patients. However, brain metastases demonstrated increased expression of several activation-specific protein markers in the PI3K/AKT pathway compared with the extracranial metastases.Conclusions: These results add to the understanding of the molecular characteristics of melanoma brain metastases and support the rationale for additional testing of the PI3K/AKT pathway as a therapeutic target in these highly aggressive tumors. Clin Cancer Res; 20(21); 5537–46. ©2014 AACR.