Christa L. Borgman

Integrative genomic and proteomic analyses identify targets for Lkb1-deficient metastatic lung tumors.

In mice, Lkb1 deletion and activation of Kras(G12D) results in lung tumors with a high penetrance of lymph node and distant metastases. We analyzed these primary and metastatic de novo lung cancers with integrated genomic and proteomic profiles, and have identified gene and phosphoprotein signatures associated with Lkb1 loss and progression to invasive and metastatic lung tumors. These studies revealed that SRC is activated in Lkb1-deficient primary and metastatic lung tumors, and that the combined inhibition of SRC, PI3K, and MEK1/2 resulted in synergistic tumor regression. These studies demonstrate that integrated genomic and proteomic analyses can be used to identify signaling pathways that may be targeted for treatment.

Inhibition of ALK, PI3K/MEK, and HSP90 in Murine Lung Adenocarcinoma Induced by EML4-ALK Fusion Oncogene

Genetic rearrangements of the anaplastic lymphoma kinase (ALK) kinase occur in 3% to 13% of non-small cell lung cancer patients and rarely coexist with KRASor EGFR mutations. To evaluate potential treatment strategies for lung cancers driven by an activated EML4-ALK chimeric oncogene, we generated a genetically engineered mouse model that phenocopies the human disease where this rearranged gene arises. In this model, the ALK kinase inhibitor TAE684 produced greater tumor regression and improved overall survival compared with carboplatin and paclitaxel, representing clinical standard of care. 18F-FDG-PET-CT scans revealed almost complete inhibition of tumor metabolic activity within 24 hours of TAE684 exposure. In contrast, combined inhibition of the PI3K/AKT and MEK/ERK1/2 pathways did not result in significant tumor regression. We identified EML4-ALK in complex with multiple cellular chaperones including HSP90. In support of a functional reliance, treatment with geldanamycin-based HSP90 inhibitors resulted in rapid degradation of EML4-ALK in vitro and substantial, albeit transient, tumor regression in vivo. Taken together, our findings define a murine model that offers a reliable platform for the preclinical comparison of combinatorial treatment approaches for lung cancer characterized by ALK rearrangement.

Hsp90 Inhibition Suppresses Mutant EGFR-T790M Signaling and Overcomes Kinase Inhibitor Resistance

The epidermal growth factor receptor (EGFR) secondary kinase domain T790M non-small cell lung cancer (NSCLC) mutation enhances receptor catalytic activity and confers resistance to the reversible tyrosine kinase inhibitors gefitinib and erlotinib. Currently, irreversible inhibitors represent the primary approach in clinical use to circumvent resistance. We show that higher concentrations of the irreversible EGFR inhibitor CL-387,785 are required to inhibit EGFR phosphorylation in T790M-expressing cells compared with EGFR mutant NSCLC cells without T790M. Additionally, CL-387,785 does not fully suppress phosphorylation of other activated receptor tyrosine kinases (RTK) in T790M-expressing cells. These deficiencies result in residual Akt and mammalian target of rapamycin (mTOR) activities. Full suppression of EGFR-mediated signaling in T790M-expressing cells requires the combination of CL-387,785 and rapamycin. In contrast, Hsp90 inhibition overcomes these limitations in vitro and depletes cells of EGFR, other RTKs, and phospho-Akt and inhibits mTOR signaling whether or not T790M is present. EGFR-T790M-expressing cells rendered resistant to CL-387,785 by a kinase switch mechanism retain sensitivity to Hsp90 inhibition. Finally, Hsp90 inhibition causes regression in murine lung adenocarcinomas driven by mutant EGFR (L858R) with or without T790M. However, efficacy in the L858R-T790M model requires a more intense treatment schedule and responses were transient. Nonetheless, these findings suggest that Hsp90 inhibitors may be effective in T790M-expressing cells and offer an alternative therapeutic strategy for this subset of lung cancers.

HER2YVMA drives rapid development of adenosquamous lung tumors in mice that are sensitive to BIBW2992 and rapamycin combination therapy

Mutations in the HER2 kinase domain have been identified in human clinical lung cancer specimens. Here we demonstrate that inducible expression of the most common HER2 mutant (HER2YVMA) in mouse lung epithelium causes invasive adenosquamous carcinomas restricted to proximal and distal bronchioles. Continuous expression of HER2YVMA is essential for tumor maintenance, suggesting a key role for HER2 in lung adenosquamous tumorigenesis. Preclinical studies assessing the in vivo effect of erlotinib, trastuzumab, BIBW2992, and/or rapamycin on HER2YVMA transgenic mice or H1781 xenografts with documented tumor burden revealed that the combination of BIBW2992 and rapamycin is the most effective treatment paradigm causing significant tumor shrinkage. Immunohistochemical analysis of lung tumors treated with BIBW2992 and rapamycin combination revealed decreased phosphorylation levels for proteins in both upstream and downstream arms of MAPK and Akt/mTOR signaling axes, indicating inhibition of these pathways. Based on these findings, clinical testing of the BIBW2992/rapamycin combination in non-small cell lung cancer patients with tumors expressing HER2 mutations is warranted.

The major lung cancer-derived mutants of ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/ ERBB2 inhibitor HKI-272.

Mutations in the ERBB2 gene were recently found in approximately 2% of primary non-small cell lung cancer (NSCLC) specimens; however, little is known about the functional consequences and the relevance to responsiveness to targeted drugs for most of these mutations. Here, we show that the major lung cancer-derived ERBB2 mutants, including the most frequent mutation, A775insYVMA, lead to oncogenic transformation in a cellular assay. Murine cells transformed with these mutants were relatively resistant to the reversible epidermal growth factor receptor (EGFR) inhibitor erlotinib, resembling the resistant phenotype found in cells carrying the homologous mutations in exon 20 of EGFR. However, the same cells were highly sensitive to the irreversible dual-specificity EGFR/ERBB2 kinase inhibitor HKI-272, as were those overexpressing wild-type ERBB2. Finally, the NSCLC cell line, Calu-3, overexpressing wild-type ERBB2 owing to a high-level amplification of the ERBB2 gene were highly sensitive to HKI-272. These results provide a rationale for treatment of patients with ERBB2-mutant or ERBB2-amplified lung tumors with HKI-272.

Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer

The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor–vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.

Early Detection of Erlotinib Treatment Response in NSCLC by 3′-Deoxy-3′-[18F]-Fluoro-L-Thymidine ([18F]FLT) Positron Emission Tomography (PET)

Background Inhibition of the epidermal growth factor receptor (EGFR) has shown clinical success in patients with advanced non-small cell lung cancer (NSCLC). Somatic mutations of EGFR were found in lung adenocarcinoma that lead to exquisite dependency on EGFR signaling; thus patients with EGFR-mutant tumors are at high chance of response to EGFR inhibitors. However, imaging approaches affording early identification of tumor response in EGFR-dependent carcinomas have so far been lacking. Methodology/Principal Findings We performed a systematic comparison of 3′-Deoxy-3′-[18F]-fluoro-L-thymidine ([18F]FLT) and 2-[18F]-fluoro-2-deoxy-D-glucose ([18F]FDG) positron emission tomography (PET) for their potential to identify response to EGFR inhibitors in a model of EGFR-dependent lung cancer early after treatment initiation. While erlotinib-sensitive tumors exhibited a striking and reproducible decrease in [18F]FLT uptake after only two days of treatment, [18F]FDG PET based imaging revealed no consistent reduction in tumor glucose uptake. In sensitive tumors, a decrease in [18F]FLT PET but not [18F]FDG PET uptake correlated with cell cycle arrest and induction of apoptosis. The reduction in [18F]FLT PET signal at day 2 translated into dramatic tumor shrinkage four days later. Furthermore, the specificity of our results is confirmed by the complete lack of [18F]FLT PET response of tumors expressing the T790M erlotinib resistance mutation of EGFR. Conclusions [18F]FLT PET enables robust identification of erlotinib response in EGFR-dependent tumors at a very early stage. [18F]FLT PET imaging may represent an appropriate method for early prediction of response to EGFR TKI treatment in patients with NSCLC.

Ganetespib (STA-9090), a Nongeldanamycin HSP90 Inhibitor, Has Potent Antitumor Activity in In Vitro and In Vivo Models of Non–Small Cell Lung Cancer

Purpose: We describe the anticancer activity of ganetespib, a novel non-geldanamycin heat shock protein 90 (HSP90) inhibitor, in non-small cell lung cancer (NSCLC) models. Experimental Design: The activity of ganetespib was compared with that of the geldanamycin 17-AAG in biochemical assays, cell lines, and xenografts, and evaluated in an ERBB2 YVMA-driven mouse lung adenocarcinoma model. Results: Ganetespib blocked the ability of HSP90 to bind to biotinylated geldanamycin and disrupted the association of HSP90 with its cochaperone, p23, more potently than 17-AAG. In genomically defined NSCLC cell lines, ganetespib caused depletion of receptor tyrosine kinases, extinguishing of downstream signaling, inhibition of proliferation and induction of apoptosis with IC50 values ranging 2 to 30 nmol/L, substantially lower than those required for 17-AAG (20–3,500 nmol/L). Ganetespib was also approximately 20-fold more potent in isogenic Ba/F3 pro-B cells rendered IL-3 independent by expression of EGFR and ERBB2 mutants. In mice bearing NCI-H1975 (EGFR L858R/T790M) xenografts, ganetespib was rapidly eliminated from plasma and normal tissues but was maintained in tumor with t1/2 58.3 hours, supporting once-weekly dosing experiments, in which ganetespib produced greater tumor growth inhibition than 17-AAG. However, after a single dose, reexpression of mutant EGFR occurred by 72 hours, correlating with reversal of antiproliferative and proapoptotic effects. Consecutive day dosing resulted in xenograft regressions, accompanied by more sustained pharmacodynamic effects. Ganetespib also showed activity against mouse lung adenocarcinomas driven by oncogenic ERBB2 YVMA. Conclusions: Ganetespib has greater potency than 17-AAG and potential efficacy against several NSCLC subsets, including those harboring EGFR or ERBB2 mutation. Clin Cancer Res; 18(18); 4973–85. ©2012 AACR.

Sunitinib Prolongs Survival in Genetically Engineered Mouse Models of Multistep Lung Carcinogenesis

Non–small cell lung cancer (NSCLC) has a poor prognosis, with substantial mortality rates even among patients diagnosed with early-stage disease. There are few effective measures to block the development or progression of NSCLC. Antiangiogenic drugs represent a new class of agents targeting multiple aspects of tumor progression, including cell proliferation, invasion, migration, and outgrowth of metastatic deposits. We tested the multitargeted angiogenesis inhibitor sunitinib in a novel endogenous mouse model of NSCLC, which expresses a conditional activating mutation in Kras with or without conditional deletion of Lkb1; both alterations are frequent in human NSCLC. We showed that daily treatment with sunitinib reduced tumor size, caused tumor necrosis, blocked tumor progression, and prolonged median survival in both the metastatic (Lkb1/Kras) and nonmetastatic (Kras) mouse models; median survival was not reached in the nonmetastatic model after 1 year. However, the incidence of local and distant metastases was similar in sunitinib-treated and untreated Lkb1/Kras mice, suggesting that prolonged survival with sunitinib in these mice was due to direct effects on primary tumor growth rather than to inhibition of metastatic progression. These collective results suggest that the use of angiogenesis inhibitors in early-stage disease for prevention of tumor development and growth may have major survival benefits in the setting of NSCLC.

Abstract C91: Pharmacodynamic analysis of the Hsp90 inhibitor STA‐9090 in a lung cancer xenograft model supports an infrequent dosing schedule in the clinic

Background: Heat shock protein 90 (Hsp90) is a molecular chaperone that is required for the stability and function of many important signal transduction proteins that regulate the growth of cancer cells. Hsp90 inhibition results in ubiquitination and proteasomal degradation of these client proteins, which include clinically validated drug targets such as BCR‐ABL, mutant EGFR, HER2, KIT and VEGFR. STA‐9090 is a novel small molecule Hsp90 inhibitor that is currently in multiple Phase 1/2 clinical trials in solid tumor and hematological malignancies. STA‐9090 is structurally unrelated to the first‐generation anasamycin Hsp90 inhibitors 17‐AAG and IPI‐504 and inhibits Hsp90 by binding to its N‐terminal ATP‐binding pocket. Although Hsp90 inhibitors such as STA‐9090 induce rapid client protein degradation, cell cycle arrest and apoptosis of cancer cells, it is possible that frequent drug dosing in the clinic may be needed to continuously maintain decreased client protein expression and avoid renewed tumor growth. To investigate this possibility, we conducted in vitro and in vivo studies using the human NCI‐H1975 non‐small cell lung cancer cell line, which expresses the Hsp90 client protein EGFRL858R/T790M, a mutationally activated and erlotinib‐resistant form of the epidermal growth factor receptor. Results: In an in vitro cytotoxicity assay using this cell line, STA‐9090 and 17‐AAG displayed IC50 values of 10 and 40 nM after 72 hr drug exposure, respectively. These results closely correlated with decreased expression of EGFRL858R/T790M and other Hsp90 client proteins. Unexpectedly, exposure to STA‐9090 for only 1 hr still resulted in an IC50 of 670 nM, suggesting that even brief drug exposure in vivo may be sufficient to affect tumor growth. Consistent with this, intravenous dosing of 125 mg/kg STA‐9090 on a 1X/week × 3 week schedule (∼80–100% of the highest non‐severely toxic dose) induced stable disease in a NCI‐H1975 xenograft model, whereas 175 mg/kg 17‐AAG resulted in progressive disease, with %T/C values of 15 and 50, respectively. Inhibition of tumor growth was correlated with decreased expression of EGFRL858R/T790M and other client proteins, and importantly, these effects persisted in tumors for 3–6 days after a single drug dose. Similarly, histological analysis of tumors indicated that STA‐9090 inhibited cell proliferation by 7‐fold and induced apoptosis by 9‐fold, with maximal effects being observed at 1–3 days after treatment. Consistent with these observations, STA‐9090 accumulated in tumors relative to normal tissues, with a tumor half‐life of 58 hr versus 3–5 hr in liver, lung and plasma, and the tumor concentration remained 140‐fold higher than the in vitro IC50 (72 hr) even 6 days after a single drug dose. Conclusions: Taken together, these results demonstrate that STA‐9090 is a highly potent Hsp90 inhibitor that selectively accumulates in tumors and induces long‐lasting client protein degradation, cell cycle arrest, increased apoptosis and tumor growth inhibition in a lung cancer xenograft model. Our results suggest that an infrequent dosing schedule may have clinical activity in cancer patients. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C91.