Manuel Sequeira

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.

Synergistic activity of the Hsp90 inhibitor ganetespib with taxanes in non-small cell lung cancer models

SummarySystemic chemotherapy using two-drug platinum-based regimens for the treatment of advanced stage non-small cell lung cancer (NSCLC) has largely reached a plateau of effectiveness. Accordingly, efforts to improve survival and quality of life outcomes have more recently focused on the use of molecularly targeted agents, either alone or in combination with standard of care therapies such as taxanes. The molecular chaperone heat shock protein 90 (Hsp90) represents an attractive candidate for therapeutic intervention, as its inhibition results in the simultaneous blockade of multiple oncogenic signaling cascades. Ganetespib is a non-ansamycin inhibitor of Hsp90 currently under clinical evaluation in a number of human malignancies, including NSCLC. Here we show that ganetespib potentiates the cytotoxic activity of the taxanes paclitaxel and docetaxel in NSCLC models. The combination of ganetespib with paclitaxel, docetaxel or another microtubule-targeted agent vincristine resulted in synergistic antiproliferative effects in the H1975 cell line in vitro. These benefits translated to improved efficacy in H1975 xenografts in vivo, with significantly enhanced tumor growth inhibition observed in combination with paclitaxel and tumor regressions seen with docetaxel. Notably, concurrent exposure to ganetespib and docetaxel improved antitumor activity in 5 of 6 NSCLC xenograft models examined. Our data suggest that the improved therapeutic indices are likely to be mechanistically multifactorial, including loss of pro-survival signaling and direct cell cycle effects resulting from Hsp90 modulation by ganetespib. Taken together, these findings provide preclinical evidence for the use of this combination to treat patients with advanced NSCLC.

FGFR3 translocations in bladder cancer: differential sensitivity to HSP90 inhibition based on drug metabolism.

Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor–resistant phenotype of FGFR3 mutant–expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity. Implications: Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors. Mol Cancer Res; 12(7); 1042–54. ©2014 AACR.

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.

The HSP90 inhibitor ganetespib has chemosensitizer and radiosensitizer activity in colorectal cancer

SummaryThe integration of targeted agents to standard cytotoxic regimens has improved outcomes for patients with colorectal cancer (CRC) over recent years; however this malignancy remains the second leading cause of cancer mortality in industrialized countries. Small molecule inhibitors of heat shock protein 90 (HSP90) are one of the most actively pursued classes of compounds for the development of new cancer therapies. Here we evaluated the activity of ganetespib, a second-generation HSP90 inhibitor, in models of CRC. Ganetespib reduced cell viability in a panel of CRC cell lines in vitro with low nanomolar potency. Mechanistically, drug treatment exerted concomitant effects on multiple oncogenic signaling pathways, cell cycle regulation, and DNA damage repair capacity to promote apoptosis. Combinations of ganetespib and low-dose ionizing radiation enhanced the radiosensitivity of HCT 116 cells and resulted in superior cytotoxic activity over either treatment alone. In vivo, the single-agent activity of ganetespib was relatively modest, suppressing HCT 116 xenograft tumor growth by approximately half. However, ganetespib significantly potentiated the antitumor efficacy of the 5-Fluorouracil (5-FU) prodrug capecitabine in HCT 116 xenografts, causing tumor regressions in a model that is intrinsically resistant to fluoropyrimidine therapy. This demonstration of combinatorial benefit afforded by an HSP90 inhibitor to a standard CRC adjuvant regimen provides an attractive new framework for the potential application of ganetespib as an investigational agent in this disease.

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.

Preclinical activity profile and therapeutic efficacy of the Hsp90 inhibitor ganetespib in triple-negative breast cancer

Purpose: Treatment options for patients with triple-negative breast cancer (TNBC) are largely limited to systemic chemotherapies, which have shown disappointing efficacy in the metastatic setting. Here, we undertook a comprehensive evaluation of the activity of ganetespib, a potent inhibitor of HSP90, in this malignancy. Experimental Design: The antitumor and antimetastatic activity of ganetespib was investigated using TNBC cell lines and xenograft models. Combinatorial drug analyses were performed with chemotherapeutic agents and concomitant effects on DNA damage and cell-cycle disruption were assessed in vitro; antitumor efficacy was assessed in vivo. Metabolic and objective tumor responses were evaluated in patients with metastatic TNBC undergoing ganetespib treatment. Results: Ganetespib simultaneously deactivated multiple oncogenic pathways to potently reduce cell viability in TNBC cell lines, and suppressed lung metastases in experimental models. Ganetespib potentiated the cytotoxic activity of doxorubicin via enhanced DNA damage and mitotic arrest, conferring superior efficacy to the doxorubicin–cyclophosphamide regimen in TNBC xenografts. Ganetespib also promoted mitotic catastrophe and apoptosis in combination with taxanes in vitro, and these effects translated to significantly improved combinatorial activity in vivo. Marked tumor shrinkage of metastatic lung and lymphatic lesions were seen in patients on ganetespib monotherapy. Conclusion: The preclinical activity profile and clinical evidence of tumor regression suggest that ganetespib offers considerable promise as a new therapeutic candidate to target TNBC. Clin Cancer Res; 20(2); 413–24. ©2013 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.

HSP90 Inhibitor–SN-38 Conjugate Strategy for Targeted Delivery of Topoisomerase I Inhibitor to Tumors

The clinical benefits of chemotherapy are commonly offset by insufficient drug exposures, narrow safety margins, and/or systemic toxicities. Over recent decades, a number of conjugate-based targeting approaches designed to overcome these limitations have been explored. Here, we report on an innovative strategy that utilizes HSP90 inhibitor–drug conjugates (HDC) for directed tumor targeting of chemotherapeutic agents. STA-12-8666 is an HDC that comprises an HSP90 inhibitor fused to SN-38, the active metabolite of irinotecan. Mechanistic analyses in vitro established that high-affinity HSP90 binding conferred by the inhibitor backbone could be exploited for conjugate accumulation within tumor cells. In vivo modeling showed that the HSP90 inhibitor moiety was required for selective retention of STA-12-8666, and this enrichment promoted extended release of active SN-38 within the tumor compartment. Indeed, controlled intratumoral payload release by STA-12-8666 contributed to a broad therapeutic window, sustained biomarker activity, and remarkable degree of efficacy and durability of response in multiple cell line and patient-derived xenograft models. Overall, STA-12-8666 has been developed as a unique HDC agent that employs a distinct mechanism of targeted drug delivery to achieve potent and sustained antitumor effects. These findings identify STA-12-8666 as a promising new candidate for evaluation as novel anticancer therapeutic. Mol Cancer Ther; 14(11); 2422–32. ©2015 AACR.