Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional complex that is activated in response to hypoxia and growth factors. HIF-1 plays a central role in tumor progression, invasion, and metastasis. Overexpression of the HIF-1A subunit has been observed in many human cancers and is associated with a poor prognostic outcome with conventional treatments. Targeting HIF-1 using novel small molecule inhibitors is, therefore, an attractive strategy for therapeutic development. We have generated U2OS human osteosarcoma cells stably expressing a luciferase reporter construct under the control of a hypoxia response element (U2OS-HRE-luc). The U2OS-HREluc cells were robustly and reproducibly sensitive to hypoxic stress in a HIF-1-dependent manner. We developed an automated U2OS-HRE-luc cell-based assay that was used in a high-throughput screen to identify compounds that inhibited HIF-1 activity induced by treatment with the hypoxia mimetic, deferoxamine mesylate. We performed a pilot screen of the National Cancer Institute Diversity Set of 2,000 compounds. We identified eight hit compounds, six of these were also identified by Rapisarda et al. in an independent hypoxia screen. However, there were two novel hit compounds, NSC-134754 and NSC-643735, that did not significantly inhibit constitutive luciferase activity in U2OS cells (U2OS-luc). We showed that both NSC-134754 and NSC-643735 significantly inhibited HIF-1 activity and HIF-1A protein induced by deferoxamine mesylate. Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1A protein induced by hypoxia and significantly inhibited Glut-1 expression. Finally, we showed that both NCS-134754 and NCS-643735 inhibited HIF-1A protein induced by insulin-like growth factor-1. Our cell-based assay approach has successfully identified novel compounds that differentially target hypoxia and/or growth factormediated induction of HIF-1A. (Cancer Res 2005; 65(11): 4918-28)

The hypoxia-inducible factor 1 (HIF-1) transcriptional complex is regulated by cellular oxygen levels and growth factors. The phosphoinosotide 3-kinase (PI-3K)-Akt/protein kinase B (PKB) pathway has been shown to regulate HIF-1 activity in response to oncogenic signals and growth factors. We assessed whether the HDM2 oncoprotein, a direct target of Akt/PKB, could regulate HIF-1␣ expression and HIF-1 activity under normoxic conditions. We found that growth factor stimulation, overexpression of Akt/PKB, or loss of PTEN resulted in enhanced expression of both HIF-1␣ and HDM2. Growth factor-mediated induction of HIF-1␣ was ablated by transient expression of a dominant negative form of Akt/PKB or by treatment with LY294002. Transient expression of HDM2 led to increased expression of HIF-1␣. Pulse-chase and cycloheximide experiments revealed that HDM2 did not significantly affect the half-life of HIF-1␣. Growth factor-induced HIF-1␣ and HDM2 proteins were localized to the nucleus, and induction of both proteins was observed in both p53؉/؉ and p53HCT116 cells to comparable levels. Importantly, insulin-like growth factor 1-induced HIF-1␣ expression was observed in p53-null mouse embryo fibroblasts (MEFs) but was significantly impaired in p53 Mdm2 doublenull MEFs, indicating a requirement for Mdm2 in this process. Finally, we showed that phosphorylation at Ser166 in HDM2 contributed in part to growth factor-mediated induction of HIF-1␣. Our study has important implications for the role of the PI-3K-Akt/PKB-HDM2 pathway in tumor progression and angiogenesis.

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