Jaime Acquaviva

Oncogenic EGFR signaling cooperates with loss of tumor suppressor gene functions in gliomagenesis

Glioblastoma multiforme (GBM) is a highly lethal brain tumor for which little treatment is available. The epidermal growth factor receptor (EGFR) signaling pathway is thought to play a crucial role in GBM pathogenesis, initiating the early stages of tumor development, sustaining tumor growth, promoting infiltration, and mediating resistance to therapy. The importance of this pathway is highlighted in the fact that EGFR is mutationally activated in over 50% of GBM tumors. Consistent with this, we show here that concomitant activation of wild-type and/or mutant (vIII) EGFR and ablation of Ink4A/Arf and PTEN tumor suppressor gene function in the adult mouse central nervous system generates a fully penetrant, rapid-onset high-grade malignant glioma phenotype with prominent pathological and molecular resemblance to GBM in humans. Studies of the activation of signaling events in these GBM tumor cells revealed notable differences between wild-type and vIII EGFR-expressing cells. We show that wild-type EGF receptor signals through its canonical pathways, whereas tumors arising from expression of mutant EGFRvIII do not use these same pathways. Our findings provide critical insights into the role of mutant EGFR signaling function in GBM tumor biology and set the stage for testing of targeted therapeutic agents in the preclinical models described herein.

The multifaceted roles of the receptor tyrosine kinase ROS in development and cancer

The proto-oncogene receptor tyrosine kinase ROS was originally discovered through the identification of oncogenic variants isolated from tumors. These discoveries spearheaded a body of work aimed at elucidating the function of this evolutionarily conserved receptor in development and cancer. Through genetic and biochemical approaches, progress in the characterization of ROS points to distinctive roles in the program of epithelial cell differentiation during the development of a variety of organs. Although substantial, these advances remain hampered by the absence of an identified ligand, making ROS one of the last two remaining orphan receptor tyrosine kinases. Recent studies on the oncogenic activation of ROS as a result of different chromosomal rearrangements found in brain and lung cancers have shed light on the molecular mechanisms underlying ROS transforming activities. ROS and its oncogenic variants therefore constitute clinically relevant targets for cancer therapeutic intervention. This review highlights the various roles that this receptor plays in multiple system networks in normalcy and disease and points to future directions towards the elucidation of ROS function in the context of ligand identification, signaling pathways and clinical applications.

Targeting KRAS-Mutant Non–Small Cell Lung Cancer with the Hsp90 Inhibitor Ganetespib

Mutant KRAS is a feature of more than 25% of non–small cell lung cancers (NSCLC) and represents one of the most prevalent oncogenic drivers in this disease. NSCLC tumors with oncogenic KRAS respond poorly to current therapies, necessitating the pursuit of new treatment strategies. Targeted inhibition of the molecular chaperone Hsp90 results in the coordinated blockade of multiple oncogenic signaling pathways in tumor cells and has thus emerged as an attractive avenue for therapeutic intervention in human malignancies. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90 currently in clinical trials for NSCLCs in a panel of lung cancer cell lines harboring a diverse spectrum of KRAS mutations. In vitro, ganetespib was potently cytotoxic in all lines, with concomitant destabilization of KRAS signaling effectors. Combinations of low-dose ganetespib with MEK or PI3K/mTOR inhibitors resulted in superior cytotoxic activity than single agents alone in a subset of mutant KRAS cells, and the antitumor efficacy of ganetespib was potentiated by cotreatment with the PI3K/mTOR inhibitor BEZ235 in A549 xenografts in vivo. At the molecular level, ganetespib suppressed activating feedback signaling loops that occurred in response to MEK and PI3K/mTOR inhibition, although this activity was not the sole determinant of combinatorial benefit. In addition, ganetespib sensitized mutant KRAS NSCLC cells to standard-of-care chemotherapeutics of the antimitotic, topoisomerase inhibitor, and alkylating agent classes. Taken together, these data underscore the promise of ganetespib as a single-agent or combination treatment in KRAS-driven lung tumors. Mol Cancer Ther; 11(12); 2633–43. ©2012 AACR.

Acquired MET expression confers resistance to EGFR inhibition in a mouse model of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which there is no cure. Overexpression of wild-type epidermal growth factor receptor (EGFR) and loss of the tumor suppressor genes Ink4a/Arf and PTEN are salient features of this deadly cancer. Surprisingly, targeted inhibition of EGFR has been clinically disappointing, demonstrating an innate ability for GBM to develop resistance. Efforts at modeling GBM in mice using wild-type EGFR have proven unsuccessful to date, hampering endeavors at understanding molecular mechanisms of therapeutic resistance. Here, we describe a unique genetically engineered mouse model of EGFR-driven gliomagenesis that uses a somatic conditional overexpression and chronic activation of wild-type EGFR in cooperation with deletions in the Ink4a/Arf and PTEN genes in adult brains. Using this model, we establish that chronic activation of wild-type EGFR with a ligand is necessary for generating tumors with histopathological and molecular characteristics of GBMs. We show that these GBMs are resistant to EGFR kinase inhibition and we define this resistance molecularly. Inhibition of EGFR kinase activity using tyrosine kinase inhibitors in GBM tumor cells generates a cytostatic response characterized by a cell cycle arrest, which is accompanied by a substantial change in global gene expression levels. We demonstrate that an important component of this pattern is the transcriptional activation of the MET receptor tyrosine kinase and that pharmacological inhibition of MET overcomes the resistance to EGFR inhibition in these cells. These findings provide important new insights into mechanisms of resistance to EGFR inhibition and suggest that inhibition of multiple targets will be necessary to provide therapeutic benefit for GBM patients.

Potent activity of the Hsp90 inhibitor ganetespib in prostate cancer cells irrespective of androgen receptor status or variant receptor expression

Androgen ablation therapy represents the first line of therapeutic intervention in men with advanced or recurrent prostate tumors. However, the incomplete efficacy and lack of durable response to this clinical strategy highlights an urgent need for alternative treatment options to improve patient outcomes. Targeting the molecular chaperone heat shock protein 90 (Hsp90) represents a potential avenue for therapeutic intervention as its inhibition results in the coordinate blockade of multiple oncogenic signaling pathways in cancer cells. Moreover, Hsp90 is essential for the stability and function of numerous client proteins, a number of which have been causally implicated in the pathogenesis of prostate cancer, including the androgen receptor (AR). Here, we examined the preclinical activity of ganetespib, a small molecule inhibitor of Hsp90, in a panel of prostate cancer cell lines. Ganetespib potently decreased viability in all lines, irrespective of their androgen sensitivity or receptor status, and more effectively than the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Interestingly, while ganetespib exposure decreased AR expression and activation, the constitutively active V7 truncated isoform of the receptor was unaffected by Hsp90 inhibition. Mechanistically, ganetespib exerted concomitant effects on mitogenic and survival pathways, as well as direct modulation of cell cycle regulators, to induce growth arrest and apoptosis. Further, ganetespib displayed robust antitumor efficacy in both AR-negative and positive xenografts, including those derived from the 22Rv1 prostate cancer cell line that co-expresses full-length and variant receptors. Together these data suggest that further investigation of ganetespib as a new therapeutic treatment for prostate cancer patients is warranted.

Overcoming Acquired BRAF Inhibitor Resistance in Melanoma via Targeted Inhibition of Hsp90 with Ganetespib

Activating BRAF kinase mutations serve as oncogenic drivers in over half of all melanomas, a feature that has been exploited in the development of new molecularly targeted approaches to treat this disease. Selective BRAFV600E inhibitors, such as vemurafenib, typically induce initial, profound tumor regressions within this group of patients; however, durable responses have been hampered by the emergence of drug resistance. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90, in melanoma lines harboring the BRAFV600E mutation. Ganetespib exposure resulted in the loss of mutant BRAF expression and depletion of mitogen-activated protein kinase and AKT signaling, resulting in greater in vitro potency and antitumor efficacy compared with targeted BRAF and MAP–ERK kinase (MEK) inhibitors. Dual targeting of Hsp90 and BRAFV600E provided combinatorial benefit in vemurafenib-sensitive melanoma cells in vitro and in vivo. Importantly, ganetespib overcame mechanisms of intrinsic and acquired resistance to vemurafenib, the latter of which was characterized by reactivation of extracellular signal-regulated kinase (ERK) signaling. Continued suppression of BRAFV600E by vemurafenib potentiated sensitivity to MEK inhibitors after acquired resistance had been established. Ganetespib treatment reduced, but not abolished, elevations in steady-state ERK activity. Profiling studies revealed that the addition of a MEK inhibitor could completely abrogate ERK reactivation in the resistant phenotype, with ganetespib displaying superior combinatorial activity over vemurafenib. Moreover, ganetespib plus the MEK inhibitor TAK-733 induced tumor regressions in vemurafenib-resistant xenografts. Overall these data highlight the potential of ganetespib as a single-agent or combination treatment in BRAFV600E-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors. Mol Cancer Ther; 13(2); 353–63. ©2014 AACR.

Targeted inhibition of Hsp90 by ganetespib is effective across a broad spectrum of breast cancer subtypes

SummaryHeat shock protein 90 (Hsp90) is a molecular chaperone essential for the stability and function of multiple cellular client proteins, a number of which have been implicated in the pathogenesis of breast cancer. Here we undertook a comprehensive evaluation of the activity of ganetespib, a selective Hsp90 inhibitor, in this malignancy. With low nanomolar potency, ganetespib reduced cell viability in a panel of hormone receptor-positive, HER2-overexpressing, triple-negative and inflammatory breast cancer cell lines in vitro. Ganetespib treatment induced a rapid and sustained destabilization of multiple client proteins and oncogenic signaling pathways and even brief exposure was sufficient to induce and maintain suppression of HER2 levels in cells driven by this receptor. Indeed, HER2-overexpressing BT-474 cells were comparatively more sensitive to ganetespib than the dual HER2/EGFR tyrosine kinase inhibitor lapatinib in three-dimensional culture. Ganetespib exposure caused pleiotropic effects in the inflammatory breast cancer line SUM149, including receptor tyrosine kinases, MAPK, AKT and mTOR signaling, transcription factors and proteins involved in cell cycle, stress and apoptotic regulation, as well as providing combinatorial benefit with lapatinib in these cells. This multimodal activity translated to potent antitumor efficacy in vivo, suppressing tumor growth in MCF-7 and MDA-MB-231 xenografts and inducing tumor regression in the BT-474 model. Thus, ganetespib potently inhibits Hsp90 leading to the degradation of multiple clinically-validated oncogenic client proteins in breast cancer cells, encompassing the broad spectrum of molecularly-defined subtypes. This preclinical activity profile suggests that ganetespib may offer considerable promise as a new therapeutic candidate for patients with advanced breast cancers.

mTOR Inhibition Potentiates HSP90 Inhibitor Activity via Cessation of HSP Synthesis

Because of their pleiotropic effects on critical oncoproteins, inhibitors of HSP90 represent a promising new class of therapeutic agents for the treatment of human cancer. However, pharmacologic inactivation of HSP90 subsequently triggers a heat shock response that may mitigate the full therapeutic benefit of these compounds. To overcome this limitation, a clinically feasible method was sought to block HSP synthesis induced by the potent HSP90 inhibitor ganetespib. An immunoassay screen of 322 late-stage or clinically approved drugs was performed to uncover compounds that could block upregulation of the stress-inducible HSP70 that results as a consequence of HSP90 blockade. Interestingly, inhibitors of the phosphoinositide 3-kinase (PI3K)/mTOR class counteracted ganetespib-induced HSP70 upregulation at both the gene and protein level by suppressing nuclear translocation of heat shock factor 1 (HSF1), the dominant transcription factor controlling cellular stress responses. This effect was conserved across multiple tumor types and was found to be regulated, in part, by mTOR-dependent translational activity. Pretreatment with cycloheximide, PI3K/mTOR inhibitor, or an inhibitor of eIF4E (a translation initiation factor and downstream effector of mTOR) all reduced ganetespib-mediated nuclear HSF1 accumulation, indicating that mTOR blockade confers a negative regulatory effect on HSF1 activity. Moreover, combined therapy regimens with mTOR or dual PI3K/mTOR inhibitors potentiated the antitumor efficacy of ganetespib in multiple in vivo models. Implications: Collectively these data identify a novel strategy to optimize the therapeutic potential of HSP90 inhibitors. Mol Cancer Res; 12(5); 703–13. ©2014 AACR.

The HSP90 inhibitor ganetespib potentiates the antitumor activity of EGFR tyrosine kinase inhibition in mutant and wild-type non-small cell lung cancer

Small molecule inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase activity, such as erlotinib and gefitinib, revolutionized therapy for non-small cell lung cancer (NSCLC) patients whose tumors harbor activating EGFR mutations. However, mechanisms to overcome the invariable development of acquired resistance to such agents, as well as realizing their full clinical potential within the context of wild-type EGFR (WT-EGFR) disease, remain to be established. Here, the antitumor efficacy of targeted EGFR tyrosine kinase inhibitors (TKIs) and the HSP90 inhibitor ganetespib, alone and in combination, were evaluated in NSCLC. Ganetespib potentiated the efficacy of erlotinib in TKI-sensitive, mutant EGFR-driven NCI-HCC827 xenograft tumors, with combination treatment causing significant tumor regressions. In erlotinib-resistant NCI-H1975 xenografts, concurrent administration of ganetespib overcame erlotinib resistance to significantly improve tumor growth inhibition. Ganetespib co-treatment also significantly enhanced antitumor responses to afatinib in the same model. In WT-EGFR cell lines, ganetespib potently reduced cell viability. In NCI-H1666 cells, ganetespib-induced loss of client protein expression, perturbation of oncogenic signaling pathways, and induction of apoptosis translated to robust single-agent activity in vivo. Dual ganetespib/erlotinib therapy induced regressions in NCI-H322 xenograft tumors, indicating that the sensitizing properties of ganetespib for erlotinib were conserved within the WT-EGFR setting. Mechanistically, combined ganetespib/erlotinib exposure stabilized EGFR protein levels in an inactive state and completely abrogated extracellular-signal-regulated kinase (ERK) and AKT signaling activity. Thus, selective HSP90 blockade by ganetespib represents a potentially important complementary strategy to targeted TKI inhibition alone for inducing substantial antitumor responses and overcoming resistance, in both the mutant and WT-EGFR settings.

Chronic Activation of Wild-Type Epidermal Growth Factor Receptor and Loss of Cdkn2a Cause Mouse Glioblastoma Formation

Glioblastoma multiforme (GBM) is characterized by overexpression of epidermal growth factor receptor (EGFR) and loss of the tumor suppressors Ink4a/Arf. Efforts at modeling GBM using wild-type EGFR in mice have proven unsuccessful. Here, we present a unique mouse model of wild-type EGFR-driven gliomagenesis. We used a combination of somatic conditional overexpression and ligand-mediated chronic activation of EGFR in cooperation with Ink4a/Arf loss in the central nervous system of adult mice to generate tumors with the histopathologic and molecular characteristics of human GBMs. Sustained, ligand-mediated activation of EGFR was necessary for gliomagenesis, functionally substantiating the clinical observation that EGFR-positive GBMs from patients express EGFR ligands. To gain a better understanding of the clinically disappointing EGFR-targeted therapies for GBM, we investigated the molecular responses to EGFR tyrosine kinase inhibitor (TKI) treatment in this model. Gefitinib treatment of primary GBM cells resulted in a robust apoptotic response, partially conveyed by mitogen-activated protein kinase (MAPK) signaling attenuation and accompanied by BIM(EL) expression. In human GBMs, loss-of-function mutations in the tumor suppressor PTEN are a common occurrence. Elimination of PTEN expression in GBM cells posttumor formation did not confer resistance to TKI treatment, showing that PTEN status in our model is not predictive. Together, these findings offer important mechanistic insights into the genetic determinants of EGFR gliomagenesis and sensitivity to TKIs and provide a robust discovery platform to better understand the molecular events that are associated with predictive markers of TKI therapy.