Cancers with specific genetic mutations are susceptible to selective kinase inhibitors. However, there is a wide spectrum of benefit among cancers harboring the same sensitizing genetic mutations. Herein, we measured apoptotic rates among cell lines sharing the same driver oncogene following treatment with the corresponding kinase inhibitor. There was a wide range of kinase inhibitor-induced apoptosis despite comparable inhibition of the target and associated downstream signaling pathways. Surprisingly, pretreatment RNA levels of the BH3-only pro-apoptotic BIM strongly predicted the capacity of EGFR, HER2, and PI3K inhibitors to induce apoptosis in EGFR-mutant, HER2-amplified, and PIK3CA-mutant cancers, respectively, but BIM levels did not predict responsiveness to standard chemotherapies. Furthermore, BIM RNA levels in EGFR-mutant lung cancer specimens predicted response and duration of clinical benefit from EGFR inhibitors. These findings suggest assessment of BIM levels in treatment-naïve tumor biopsies may indicate the degree of benefit from single-agent kinase inhibitors in multiple oncogene-addiction paradigms. Significance: In several oncogene-addiction paradigms, assessment of BIM RNA levels identifies those cancers that fail to have substantial apoptotic responses to kinase inhibitors. BIM RNA levels may be assessed in diagnostic cancer specimens to predict which patients will receive less benefit from single-agent kinase inhibitors. Cancer Discovery: 1(4); 352–65. ©2011 AACR. Read the Commentary on this article by Yoshida and Haura, p. 289 This article is highlighted in the In This Issue feature, p. 275

Current anti-VEGF therapies for colorectal cancer (CRC) provide limited survival benefit, as tumors rapidly develop resistance to these agents. Here, we have uncovered an immunosuppressive role for nonclassical Ly6C^lo monocytes that mediates resistance to anti-VEGFR2 treatment. We found that the chemokine CX3CL1 was upregulated in both human and murine tumors following VEGF signaling blockade, resulting in recruitment of CX3CR1⁺Ly6C^lo monocytes into the tumor. We also found that treatment with VEGFA reduced expression of CX3CL1 in endothelial cells in vitro. Intravital microscopy revealed that CX3CR1 is critical for Ly6C^lo monocyte transmigration across the endothelium in murine CRC tumors. Moreover, Ly6C^lo monocytes recruit Ly6G⁺ neutrophils via CXCL5 and produce IL-10, which inhibits adaptive immunity. Preventing Ly6C^lo monocyte or Ly6G⁺ neutrophil infiltration into tumors enhanced inhibition of tumor growth with anti-VEGFR2 therapy. Furthermore, a gene therapy using a nanoparticle formulated with an siRNA against CX3CL1 reduced Ly6C^lo monocyte recruitment and improved outcome of anti-VEGFR2 therapy in mouse CRCs. Our study unveils an immunosuppressive function of Ly6C^lo monocytes that, to our knowledge, has yet to be reported in any context. We also reveal molecular mechanisms underlying antiangiogenic treatment resistance, suggesting potential immunomodulatory strategies to enhance the long-term clinical outcome of anti-VEGF therapies.

Brain metastases are a serious obstacle in the treatment of patients withhuman epidermal growth factor receptor-2(HER2)–amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model ofHER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti–VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment ofHER2-amplified breast cancer brain metastases.