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T.F. Burns
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MINI 21 - Novel Targets (ID 133)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:B.P. Levy, D.S. Tan
- Coordinates: 9/08/2015, 16:45 - 18:15, Mile High Ballroom 2a-3b
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MINI21.12 - Identification of a First in Class TWIST1 Inhibitor with Activity in KRAS Mutant NSCLC (ID 1616)
16:45 - 18:15 | Author(s): T.F. Burns
- Abstract
- Presentation
Background:
Although a large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations, little progress has been made in the treatment of patients with the most common driver mutation, mutant KRAS. We previously demonstrated that the basic helix-loop-helix transcription factor, Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in mouse models, and that inhibition of Twist1 in murine models and KRAS mutant NSCLC cell lines led to oncogene-induced senescence (OIS) and in some cases, apoptosis. Therefore, targeting the TWIST1 pathway represents an exciting and novel therapeutic strategy which may have a significant clinical impact.
Methods:
We used gene expression profiles from KRAS mutant human NSCLC cell lines following shRNA-mediated TWIST1 knockdown to perform connectivity map (CMAP) analysis to identify pharmacologic inhibitors of TWIST1. Growth inhibition was determined through the colony formation and MTS assays. Apoptosis (cl-PARP, active anti-C3) and OIS (SA-β-Gal) was assessed. Genetic (shRNA) and pharmacologic inhibition of the TWIST1-E2A pathway was performed. Lung tumor burden as well as levels of TWIST1 protein, apoptosis and proliferation were measured after treatment with harmine in the CCSP-rtTA/tetO-KrasG12D/Twist1-tetO7-luc(CRT) mice.
Results:
We found that several of our CMAP compounds had significant growth inhibitory effects in NSCLC cell lines. Interestingly, a family of related harmala alkaloids including harmine ranked highly in our CMAP analysis. We observed that harmine could inhibit growth in KRAS mutant NSCLC cell lines through the induction of OIS or apoptosis and phenocopied genetic inhibition of TWIST1. Remarkably, harmine treatment led to TWIST1 protein degradation as well as degradation of its binding partners, the E2A proteins, E12/E47. Furthermore, the growth inhibitory effects of the harmala alkaloids correlated with the ability to degrade TWIST1 and were independent of its ability to inhibit the DYRK kinases. In addition, we demonstrated that heterodimer formation of TWIST1/E12/E47 resulted in a reciprocal stabilization of each binding partner and that E12/E47 are required for TWIST1 mediated suppression of OIS and apoptosis. Importantly, we found that harmine preferential targets the TWIST1-E12 heterodimer for degradation and the growth inhibitory effects of harmine are in due in at least part to the ability to inhibit the TWIST1/E12/E47 heterodimer as overexpression of the E2A proteins can suppress harmine induced cytotoxicity. Finally, we have demonstrated that harmine treatment lead to Twist1 protein degradation and tumor growth inhibition in our Kras[G12D]/Twist1 murine model of lung adenocarcinoma. We are currently testing and designing structure analogs of the initial candidate agents to develop more specific and potent inhibitors of TWIST1.
Conclusion:
We have identified a novel TWIST1 inhibitor harmine that induces degradation of TWIST1 and its binding partners, E12/E47 and inhibits the growth of KRAS mutant NSCLC both in vitro and in vivo. Therefore, we believe that targeting the TWIST1-E2A pathway would be an effective therapeutic strategy. Since TWIST1 is essential not only for KRAS mutant NSCLC but more broadly for oncogene driven NSCLC, the development of this novel class of TWIST1 inhibitors could have a significant clinical impact.
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MINI 30 - New Kinase Targets (ID 157)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Treatment of Advanced Diseases - NSCLC
- Presentations: 1
- Moderators:K. Park, M. Villalona
- Coordinates: 9/09/2015, 18:30 - 20:00, Four Seasons Ballroom F3+F4
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MINI30.02 - Phase II Study of Defactinib, VS-6063, a Focal Adhesion Kinase (FAK) Inhibitor, in Patients with KRAS Mutant Non-Small Cell Lung Cancer (NSCLC) (ID 2875)
18:30 - 20:00 | Author(s): T.F. Burns
- Abstract
- Presentation
Background:
KRAS mutations, which occur in approximately 30% of lung adenocarcinoma cases, represent a major unmet clinical need in thoracic oncology. Preclinical studies have demonstrated that KRAS mutant NSCLC cell lines and xenografts with additional alterations in either p53 or INK4a/Arf (CDKN2A) are sensitive to FAK inhibition. Defactinib (VS-6063) is a selective oral inhibitor of FAK. This trial examined the effect of FAK inhibition in patients with KRAS mutant NSCLC and various permutations of p53 and CDKN2A alterations.
Methods:
This multi-center, non-randomized, open-label, multi-cohort trial enrolled patients with advanced KRAS mutant NSCLC who had received at least one prior (platinum-based chemotherapy doublet) line of therapy. The primary endpoint was progression-free survival (PFS) at 12 weeks. Patients were enrolled into one of four cohorts defined by INK4a/Arf and p53 status. In all cohorts, patients received defactinib 400 mg orally BID until disease progression.
Results:
Fifty-three patients with KRAS mutant NSCLC were enrolled across 9 US sites as of the data cut-off date (13-Mar-2015). Forty-seven patients were enrolled to one of the four molecularly defined cohorts. The median age was 62 years (range 33-80); 48% were female. The median number of prior lines of therapy was 3 (range 1-8) 15 (28%) pts met the 12 week PFS endpoint, with one patient achieving a PR. Median PFS was 46 days (range 12-205 days). Eight patients remained on study as of the data cut-off date. Clinical efficacy did not correlate with secondary mutation status across this KRAS mutant population. Adverse events considered at least possibly related to defactinib were experienced by 35 pts (76%). The majority of these were grade 1 or 2. 11 patients (24%) experienced at least possibly related grade 3-5 events, including 2 grade 5 respiratory failure events. Underlying disease was a confounding factor in many pts. The most commonly reported treatment emergent adverse events of any grade were fatigue (24%) and increased bilirubin (24%).
Conclusion:
In pretreated pts with KRAS mutant NSCLC defactinib demonstrates promising clinical activity with disease control rates comparable to other molecularly targeted agents for this pt population. Defactinib was generally well tolerated. Further development is warranted. Clinical trial: NCT01778803.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-101 - Ganetespib Resistance in KRAS Mutant NSCLC Is Mediated through Reactivation of the RAF/MEK/ERK and PI3K/MTOR Pathways (ID 1626)
09:30 - 17:00 | Author(s): T.F. Burns
- Abstract
Background:
One third of all malignancies and approximately 25% of non-small cell lung cancer (NSCLC) patients have KRAS mutations which leads to the activation of several growth regulatory signaling pathways including RAF/MEK/ERK and PI3K/AKT/MTOR. Unfortunately, there are no current therapies targeting this critical oncogene. Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability of ‘client’ oncoproteins, many of which are effectors of KRAS. Unfortunately, limited efficacy was observed in early clinical studies of single agent HSP90 inhibitors (HSP90i) in KRAS mutant NSCLC. Here, we examined the mechanism(s) of acquired resistance to ganetespib, a Phase 3 HSP90i, in KRAS mutant NSCLC to develop rationale combinations with ganetespib.
Methods:
Growth inhibition was determined through the colony formation and MTS assays. Ganetespib resistant (GR)-KRAS mutant NSCLC cell lines were derived to identify resistance mechanism(s). Flow-cytometry was performed to generate cell-cycle profiles. Genetic (shRNA) and pharmacologic inhibition of candidate mediators of resistance was performed.
Results:
Ganetespib was cytotoxic in a panel of KRAS mutant NSCLC cell lines and decreased expression and activity of both RAF/MEK/ERK and PI3K/AKT/MTOR pathways. In order to identify the mechanisms of ganetespib resistance in KRAS mutant NSCLC, we derived three KRAS mutant NSCLC ganetespib resistant (GR) cell lines. GR cells were cross-resistant to a first generation HSP90i, 17-AAG, suggesting that altered metabolism of ganetespib is unlikely to explain this resistance. Moreover, the ganetespib-induced G~2~/M checkpoint arrest observed in A549 parental cells was significantly diminished in A549-GR cells. These results suggest that bypass of this checkpoint may contribute to the observed ganetespib resistance. Furthermore, we demonstrated that GR cells were cross-resistant to docetaxel, an anti-microtubule agent. In addition, expression and activity of the PI3K/AKT/MTOR pathway members as well as the RAF/MEK/ERK pathway members were significantly increased suggesting that reactivation of these pathways may be responsible for the observed resistance. To test this hypothesis, we treated parental and GR cells with inhibitors of the PI3K/AKT/MTOR pathway (dual PI3K/mTOR inhibitor, BEZ235 and PI3K inhibitor PX866) or the RAF/MEK/ERF pathway (ERK inhibitor, SCH772984). Remarkably, GR cells were more sensitive to these inhibitors compared to the parental ones suggesting that the acquired ganetespib resistance lead to increased dependence on the both RAF/MEK/ERK and PI3K/MTOR pathways. Interestingly, the expression/activity of the key ERK and PDK1 substrate and activator of the PI3K/MTOR pathway, p90 ribosomal S6 kinase (RSK) was strikingly increased in the GR cells. Since RSK has been implicated as a key mediator of crosstalk between these two pathways, as well as in promoting G2/M progression, we examine the effect of genetic (shRNA) or pharmacologic (BI-D1870 and SL0101) inhibition of RSK in two GR cell lines. Remarkably, the GR cells showed increased dependency on RSK activity compared to the parental cell lines.
Conclusion:
These data suggests that the combination of inhibitors for HSP90 and PI3K/mTOR or a RSK inhibitor may prevent ganetespib resistance and/or help overcome the resistance after single agent treatment, providing the preclinical rationale for our planned Phase I/II trial of the combination of ganetespib and a dual PI3K/MTOR inhibitor in KRAS mutant NSCLC.
