PF-03814735 is a novel, reversible inhibitor of Aurora kinases A and B that finished a phase I clinical trial for the treatment of advanced solid tumors. To find predictive biomarkers of drug sensitivity, we screened a diverse panel of 87 cancer cell lines for growth inhibition upon PF-03814735 treatment. Small cell lung cancer (SCLC) and, to a lesser extent, colon cancer lines were very sensitive to PF-03814735. The status of the Myc gene family and retinoblastoma pathway members significantly correlated with the efficacy of PF-03814735. Whereas RB1 inactivation, intact CDKN2A/p16, and normal CCND1/Cyclin D1 status are hallmarks of SCLC, activation or amplification of any of the three Myc genes (MYC, MYCL1, and MYCN) clearly differentiated cell line sensitivity within the SCLC panel. By contrast, we found that expression of Aurora A and B were weak predictors of response. We observed a decrease in histone H3 phosphorylation and polyploidization of sensitive lines, consistent with the phenotype of Aurora B inhibition. In vivo experiments with two SCLC xenograft models confirmed the sensitivity of Myc gene-driven models to PF-03814735 and a possible schedule dependence of MYC/c-Myc-driven tumors. Altogether our results suggest that SCLC and other malignancies driven by the Myc family genes may be suitable indications for treatment by Aurora B kinase inhibitors.

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Metcycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or smallmolecule inhibition leads to de-repression of RXRa resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

Figitumumab (CP-751,871), a potent and fully human monoclonal anti-insulin-like growth factor 1 receptor (IGF1R) antibody, has been investigated in clinical trials of several solid tumors. To identify biomarkers of sensitivity and resistance to figitumumab, its in vitro antiproliferative activity was analyzed in a panel of 93 cancer cell lines by combining in vitro screens with extensive molecular profiling of genomic aberrations. Overall response was bimodal and the majority of cell lines were resistant to figitumumab. Nine of 15 sensitive cell lines were derived from colon cancers. Correlations between genomic characteristics of cancer cell lines with figitumumab antiproliferative activity revealed that components of the IGF pathway, including IRS2 (insulin receptor substrate 2) and IGFBP5 (IGF-binding protein 5), played a pivotal role in determining the sensitivity of tumors to single-agent figitumumab. Tissue-specific differences among the top predictive genes highlight the need for tumor-specific patient selection strategies. For the first time, we report that alteration or expression of the MYB oncogene is associated with sensitivity to IGF1R inhibitors. MYB is dysregulated in hematologic and epithelial tumors, and IGF1R inhibition may represent a novel therapeutic opportunity. Although growth inhibitory activity with single-agent figitumumab was relatively rare, nine combinations comprising figitumumab plus chemotherapeutic agents or other targeted agents exhibited properties of synergy. Inhibitors of the ERBB family were frequently synergistic and potential biomarkers of drug synergy were identified. Several biomarkers of antiproliferative activity of figitumumab both alone and in combination with other therapies may inform the design of clinical trials evaluating IGF1R inhibitors.

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The phosphatidylinositol 3-kinase/mechanistic target of rapamycin (PI3K/mTOR) pathway and the mitogen-activated protein kinase (MAPK) signaling cascades are central facilitators of cellular growth, proliferation, survival and motility within a variety of tumor lineages. To date, several ongoing oncology clinical trials are currently evaluating the benefit of combining PI3K with MEK inhibition. While this dual combination is well founded pre-clinically, investigating potential mechanisms of resistance and identifying novel biological signatures of sensitivity is of great clinical importance. We sought to establish a model of acquired resistance by evaluating the co-treatment of a colorectal carcinoma model with inhibitors of PI3K/mTOR inhibitor (PF-04691502) and MEK (PD-0325901). We can associate the onset of acquired resistance in this model to re-engagement of signaling within the PI3K/mTOR pathway correlative to a loss of PTEN protein expression , relative to parental cells. In addition, next-generation sequencing (NSG) comparing parental versus resistant cells identified a unique somatic “gatekeeper” mutation located in the kinase domain of mTOR. Furthermore, a cell proliferation screen evaluating potential cross resistance was analyzed using a PI3K/mTOR inhibitor with a chemical scaffold distinct from PF-502 (PF-05212384). Treatment of PF-384 as a single agent (IC50 ∼150nM) circumvented the acquired resistance derived from the combination of PF-502+ PD-901 (IC50: ∼2uM and ∼334nM respectfully). Inhibition of growth observed with PF-384 correlated with rescuing of PI3K/mTOR signaling as measured by substrate phosphorylation readouts. Finally, co-crystal structure modeling of mTOR was used to evaluate the potential interaction of PF-502 within the ATP binding pocket and we confirm that this interaction is hindered due to the point mutation identified at mTOR. Furthermore, co-crystal modeling of mTOR with PF-384 illustrates an independence and lack of PF-384 binding to the mutated site and thus substantiates a mechanistic explanation for overcoming PF-502 acquired resistance. In summary, we have identified and characterized a novel mechanism of resistance to the combined treatment of a Pyrimidinone class PI3K/mTOR inhibitor in addition to a MEK inhibitor, and have shown that rescuing of this resistance can be achieved through the use of a Triazine class PI3K/mTOR clinical candidate. Citation Format: Scott J. Garza, Paul Lira, Stephen D. Huang, Hengmiao Cheng, Stephen G. Dann, Todd L. VanArsdale, Valeria Fantin, James Christensen, Julie LC Kan. Identifying a mechanism of acquired resistance to the combined inhibition of PI3K/mTOR and MEK in colorectal carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3713. doi:10.1158/1538-7445.AM2014-3713

The PI3K pathway plays a role in key cellular functions that include regulation of cell growth, proliferation, survival, angiogenesis, and motility. PI3K/AKT pathway aberrations are common in cancer comprising PTEN loss of function through mutations, deletion, and methylation events and gain of function at the PIK3CA locus including mutations and amplification events resulting in deregulation of this pathway. Thus, pharmacological intervention of this pathway should impact cellular functions central to survival of cancer cells. PIK3CA mutations/amplification and PTEN loss of heterozygosity have been reported to occur in approximately 35-40% and 20% of breast cancer, respectively and suggest this patient population could benefit from treatment using a small molecule inhibitor that targets the PI3K pathway. PF-04691502 which is a potent inhibitor of all PI3K isoforms and mTOR (TORC1 and TORC2) presently in Phase 1 trials was evaluated for its growth inhibitory or cytoreductive activity over a panel of 30 breast cancer cell lines. PF-04691502 had robust <300nM (IC50%) antiproliferative activity across the panel of breast cell lines. We observed a trend whereby cell lines that were hormone receptor (ER and/or PR) and HER2 negative (triple negative) were least sensitive and cell lines that were HER2(+) and/or hormone receptor positive (ER and/or PR) were more sensitive to PF-04691502. These studies were extended in vivo with PF-04691502 as a single agent and in combination in a HER2, ER and PR positive xenograft model with robust tumor growth inhibition in combination with docetaxel or with the pan-HER inhibitor, PF-00299804. In a xenograft that was triple negative, administration of single agent PF-4691502 at the MTD resulted in 60% tumor growth inhibition. In conclusion, our findings suggest that the ER/PR and/or HER2 positive breast cancer segment may particularly benefit from PI3K/mTOR inhibitor based treatment regimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-302.

The identification of patient populations predicted to derive clinical benefit from systemic treatment regimens is of critical importance for development of targeted therapies. Collective clinical data available for the Aurora kinase inhibitor class of targeted therapies has indicated that broad single agent clinical activity is not readily apparent. We screened a panel of established lung (mostly non-small cell) and colon cell lines for growth inhibitory sensitivity to PF-03814735, an Aurora family kinase inhibitor, revealing a potential correlation with Myc family amplification or expression. The Aurora kinases A and B have been shown to be functionally linked with Myc family oncoproteins in a number of tumor types. Myc family gene amplification events have been reported in about 30% of small cell lung cancer (SCLC) primary tumors and around 50% of cell lines established from SCLC. We next screened a panel of around 20 SCLC lines for sensitivity to PF-03814735 and its relationship to Myc family gene copy number and gene expression levels. Sensitivity to PF-03814735 in vitro was strongly correlated with amplification events in at least one of the Myc family genes (c-Myc, L-Myc, N-Myc) as well as mRNA expression levels of those genes. Myc family expression demonstrated a significant correlation with gene copy number. Follow up studies to evaluate antitumor efficacy in two SCLC xenograft models, H82 (c-Myc) and H69 (N-Myc), indicated significant tumor growth inhibition by PF-03814735. However, the SCLC models were not appreciably more sensitive to PF-03814735 than several non-Myc amplified tumors studied previously (HCT-116, COLO-205, MDA-MB-231) indicating that further study of optimal dose schedule and/or the molecular basis of sensitivity is warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2615.

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