Inhibitor TAK-733 Research Articles

The dual mTOR kinase inhibitor TAK228 inhibits tumorigenicity and enhances radiosensitization in diffuse intrinsic pontine glioma

Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. Primary DIPG tumors harbor a number of mutations including alterations in PTEN, AKT, and PI3K and exhibit activation of mammalian Target of Rapamycin Complex 1 and 2 (mTORC1/2). mTORC1/2 regulate protein translation, cell growth, survival, invasion, and metabolism. Pharmacological inhibition of mTORC1 is minimally effective in DIPG. However, the activity of dual TORC kinase inhibitors has not been examined in this tumor type.Nanomolar levels of the mTORC1/2 inhibitor TAK228 reduced expression of p-AKTS473 and p-S6S240/244 and suppressed the growth of DIPG lines JHH-DIPG1, SF7761, and SU-DIPG-XIII. TAK228 induced apoptosis in DIPG cells and cooperated with radiation to further block proliferation and enhance apoptosis.TAK228 monotherapy inhibited the tumorigenicity of a murine orthotopic model of DIPG, more than doubling median survival (p = 0.0017) versus vehicle. We conclude that dual mTOR inhibition is a promising potential candidate for DIPG treatment.

Abstract OT2-01-21: Phase 2 study of investigational TORC1/2 inhibitor TAK-228 with fulvestrant in women with ER-positive/HER2-negative advanced or metastatic breast cancer that has progressed during or after aromatase inhibitor therapy

Abstract Standard therapy for ER-positive tumors in the adjuvant and metastatic settings is antiestrogen therapy, including aromatase inhibitors (AI); however, resistance is common. These tumors may respond to alternative second-line anti-estrogen therapies such as fulvestrant but response durations are often short. Preclinical and clinical studies suggest that simultaneous inhibition of ER and PI3K/mTOR could prevent/delay the emergence of hormone-independent cancer cells thereby improving patient (pt) outcomes. This study will test whether fulvestrant plus TAK-228, a dual TORC1/2 inhibitor, can overcome endocrine therapy resistance in ER-positive breast cancer. This is an open-label, randomized, 3-arm, phase 2 study of continuous once-daily TAK-228 (oral, 4 mg) or once-weekly TAK-228 (oral, 30 mg) plus fulvestrant (500 mg intramuscularly on d1 and d15 of cycle 1 [loading regimen] then d1 of each subsequent 28-d cycle), compared with fulvestrant alone, in pts with advanced or metastatic ER-positive/HER2-negative breast cancer that has progressed during/after AI therapy. Pts will be randomized 1:1:1 to the 3 arms and stratified at randomization via presence or absence of visceral metastasis, prior hormonal therapy sensitivity, and prior exposure to CDK 4/6 inhibitors. Pts will receive study drug(s) until progressive disease (PD), unacceptable toxicity, or consent withdrawal. Postmenopausal women ≥18 yrs old with local histological confirmation of ER-positive/HER2-negative metastatic or advanced breast cancer; with measureable disease; ECOG status 0–1; PD during/after prior AI therapy (defined as progression ≤12 mos after discontinuing adjuvant therapy or ≤1 mo after discontinuation in the metastatic setting) and adequate organ function are eligible. Exclusion criteria include prior therapy with mTOR inhibitors, PI3K inhibitors, dual PI3K-mTOR inhibitors, AKT inhibitors, or fulvestrant; prior treatment with >1 line of chemotherapy for metastatic breast cancer; experienced recurrent or progressive disease on >2 endocrine therapies for metastatic breast cancer; or significant previous/existing cardiac conditions. This study aims to determine the efficacy (primary endpoint: PFS; secondary endpoints: OS, TTP, ORR), safety and tolerability of daily and weekly TAK-228 plus fulvestrant compared with fulvestrant alone. The distribution of PFS will be analyzed via the Kaplan-Meier method. The primary hypothesis (TAK-228 plus fulvestrant can improve median PFS to 8 mos [hazard ratio, HR 0.5] vs fulvestrant-alone median PFS of 4 mos) is to be tested at the 0.10 significance level (2-sided; dropout rate 10%). A total of 72 PFS events are needed for each pair-wise comparison; p-values from a stratified log-rank test and HRs will be presented. The safety profile and clinical laboratory parameters (and/or change from baseline) for all scheduled measurements over time will be summarized by descriptive statistics. Approximately 153 pts (51 pts per arm) will be enrolled from approximately 55 study centers in North America and Spain; to date, no pts have been enrolled. For more information, please contact Michelle Kneissl at michelle.kneissl@takeda.com. Citation Format: García-Sáenz JA, Carrasco E, Kneissl ML, Zohren F, Martin M. Phase 2 study of investigational TORC1/2 inhibitor TAK-228 with fulvestrant in women with ER-positive/HER2-negative advanced or metastatic breast cancer that has progressed during or after aromatase inhibitor therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT2-01-21.

377P - Phase 1 study of investigational oral mTORC1/2 inhibitor TAK-228 (MLN0128): Tolerability and food effects of a milled formulation in patients (pts) with advanced solid tumors

377P - Phase 1 study of investigational oral mTORC1/2 inhibitor TAK-228 (MLN0128): Tolerability and food effects of a milled formulation in patients (pts) with advanced solid tumors

Design and synthesis of N-(4-aminopyridin-2-yl)amides as B-RafV600E inhibitors

B-RafV600E was an effective target for the treatment of human cancers. Based on a pan-Raf inhibitor TAK-632, a series of N-(4-aminopyridin-2-yl)amide derivatives were designed as novel B-RafV600E inhibitors. Detailed structure–activity studies of the compounds revealed that most of the compounds displayed potent enzymatic activity against B-RafV600E, and good selectivity over B-RafWT. One of the most promising compound 4l exhibited potent inhibitory activity with an IC50 value of 38nM for B-rafV600E, and displayed antiproliferative activities against colo205 and HT29 cells with IC50 values of 0.136 and 0.094μM, respectively. It also displayed good selectivity on both enzymatic and cellular assays over B-RafWT. These inhibitors may serve as lead compounds for further developing novel B-RafV600E inhibitors as anticancer drugs.

Antitumor activity of a potent MEK inhibitor, TAK-733, against colorectal cancer cell lines and patient derived xenografts.

CRC is a significant cause of cancer mortality, and new therapies are needed for patients with advanced disease. TAK-733 is a highly potent and selective investigational novel MEK allosteric site inhibitor. In a preclinical study of TAK-733, a panel of CRC cell lines were exposed to varying concentrations of the agent for 72 hours followed by a sulforhodamine B assay. Twenty patient-derived colorectal cancer xenografts were then treated with TAK-733 in vivo. Tumor growth inhibition index (TGII) was assessed to evaluate the sensitivity of the CRC explants to TAK-733 while linear regression was utilized to investigate the predictive effects of genotype on the TGII of explants. Fifty-four CRC cell lines were exposed to TAK-733, while 42 cell lines were deemed sensitive across a broad range of mutations. Eighty-two percent of the cell lines within the sensitive subset were BRAF or KRAS/NRAS mutant, whereas 80% of the cell lines within the sensitive subset were PIK3CA WT. Twenty patient-derived human tumor CRC explants were then treated with TAK-733. In total, 15 primary human tumor explants were found to be sensitive to TAK-733 (TGII ≤ 20%), including 9 primary human tumor explants that exhibited tumor regression (TGII > 100%). Explants with a BRAF/KRAS/NRAS mutant and PIK3CA wild-type genotype demonstrated increased sensitivity to TAK-733 with a median TGII of -6%. MEK-response gene signatures also correlated with responsiveness to TAK-733 in KRAS-mutant CRC. The MEK inhibitor TAK-733 demonstrated robust antitumor activity against CRC cell lines and patient-derived tumor explants. While the preclinical activity observed in this study was considerable, single-agent efficacy in the clinic has been limited in CRC, supporting the use of these models in an iterative manner to elucidate resistance mechanisms that can guide rational combination strategies.

Antitumor activity of the MEK inhibitor TAK-733 against melanoma cell lines and patient-derived tumor explants.

The goal of this study was to investigate the activity of the selective MEK1/2 inhibitor TAK-733 in both melanoma cell lines and patient-derived melanoma xenograft models. In vitro cell proliferation assays using the sulforhodamine B assay were conducted to determine TAK-733 potency and melanoma responsiveness. In vivo murine modeling with eleven patient-derived melanoma explants evaluated daily dosing of TAK-733 at 25 or 10 mg/kg. Immunoblotting was performed to evaluate on-target activity and downstream inhibition by TAK-733 in both in vitro and in vivo studies. TAK-733 demonstrated broad activity in most melanoma cell lines with relative resistance observed at IC50 > 0.1 μmol/L in vitro. TAK-733 also exhibited activity in 10 out of 11 patient-derived explants with tumor growth inhibition ranging from 0% to 100% (P < 0.001-0.03). Interestingly, BRAF(V600E) and NRAS mutational status did not correlate with responsiveness to TAK-733. Pharmacodynamically, pERK was suppressed in sensitive cell lines and tumor explants, confirming TAK-733-mediated inhibition of MEK1/2, although the demonstration of similar effects in the relatively resistant cell lines and tumor explants suggests that escape pathways are contributing to melanoma survival and proliferation. These data demonstrate that TAK-733 exhibits robust tumor growth inhibition and regression against human melanoma cell lines and patient-derived xenograft models, suggesting that further clinical development in melanoma is of scientific interest. Particularly interesting is the activity in BRAF wild-type models, where current approved therapy such as vemurafenib has been reported not to be active.

Abstract 4247: Characterization of the selective pan-RAF inhibitor TAK-632 with antitumor activity in BRAF inhibitor-resistant melanoma

Abstract Melanoma is one of the deadliest and most aggressive forms of skin cancer, arising from the malignant transformation of pigment-producing cells, melanocytes. The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells and somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, it has been reported that the BRAF inhibitors vemurafenib and dabrafenib showed high response rates and improved overall survival in melanomas with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS mutant melanoma) are clinical concerns. In this preclinical study, we describe the biological characterization of TAK-632 as a potent and selective pan-RAF inhibitor that suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. We used both genetic and chemical approaches to investigate the dependence of NRAS-mutated melanoma and BRAF inhibitor-resistant BRAF mutant melanoma cells on RAF. Our analysis reveals that the MAPK pathway and proliferation of these cells is highly dependent on RAF. Such dependence was not observed in several RAS/RAF-wild type and KRAS-mutated cells. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. Furthermore, we demonstrate that the combination of TAK-632 and various MAPK kinase (MEK) inhibitors exhibits synergistic anti-proliferative effects on these cells. Our findings indicate that TAK-632 has favorable characteristics in terms of suppressing the proliferation of NRAS mutant melanoma and BRAF inhibitor-resistant BRAF mutant melanoma cells. Citation Format: Akito Nakamura, Takeo Arita, Shuntarou Tsuchiya, Jill Donelan, Jouhara Chouitar, Elizabeth Carideo, Katherine Galvin, Masanori Okaniwa, Tomoyasu Ishikawa, Sei Yoshida. Characterization of the selective pan-RAF inhibitor TAK-632 with antitumor activity in BRAF inhibitor-resistant melanoma. [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 4247. doi:10.1158/1538-7445.AM2014-4247

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 BRAF(V600E) 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 BRAF(V600E) 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 BRAF(V600E) 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 BRAF(V600E) 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 BRAF(V600E)-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors.

Antitumor Activity of the Selective Pan-RAF Inhibitor TAK-632 in BRAF Inhibitor-Resistant Melanoma

The mitogen-activated protein kinase (MAPK) pathway is particularly important for the survival and proliferation of melanoma cells. Somatic mutations in BRAF and NRAS are frequently observed in melanoma. Recently, the BRAF inhibitors vemurafenib and dabrafenib have emerged as promising agents for the treatment of melanoma patients with BRAF-activating mutations. However, as BRAF inhibitors induce RAF paradoxical activation via RAF dimerization in BRAF wild-type cells, rapid emergence of acquired resistance and secondary skin tumors as well as presence of few effective treatment options for melanoma bearing wild-type BRAF (including NRAS-mutant melanoma) are clinical concerns. Here, we demonstrate that the selective pan-RAF inhibitor TAK-632 suppresses RAF activity in BRAF wild-type cells with minimal RAF paradoxical activation. Our analysis using RNAi and TAK-632 in preclinical models reveals that the MAPK pathway of NRAS-mutated melanoma cells is highly dependent on RAF. We also show that TAK-632 induces RAF dimerization but inhibits the kinase activity of the RAF dimer, probably because of its slow dissociation from RAF. As a result, TAK-632 demonstrates potent antiproliferative effects both on NRAS-mutated melanoma cells and BRAF-mutated melanoma cells with acquired resistance to BRAF inhibitors through NRAS mutation or BRAF truncation. Furthermore, we demonstrate that the combination of TAK-632 and the MAPK kinase (MEK) inhibitor TAK-733 exhibits synergistic antiproliferative effects on these cells. Our findings characterize the unique features of TAK-632 as a pan-RAF inhibitor and provide rationale for its further investigation in NRAS-mutated melanoma and a subset of BRAF-mutated melanomas refractory to BRAF inhibitors.

Abstract C146: Combination treatment with the investigational RAF kinase inhibitor MLN2480 and the investigational MEK kinase inhibitor TAK-733 inhibits the growth of BRAF mutant and RAS mutant preclinical models of melanoma and CRC.

Abstract Background: RAS mutant melanoma and colorectal cancer represent areas of significant unmet medical need. MLN2480 is an investigational class II RAF kinase inhibitor and TAK-733 is an investigational allosteric MEK kinase inhibitor; each of which is the subject of a single agent phase I clinical trial. The present studies have characterized the combination activity of these agents in BRAF mutant and RAS mutant preclinical models of melanoma and colorectal cancer. Methods: Combination effects of MLN2480 and TAK-733 on cell viability were studied using an ATP-based cell viability assay across a panel of BRAF and RAS mutant melanoma and CRC cell lines. Western blot analysis was used to compare effects on MAPK pathway signaling and response markers in cell lines showing a range of sensitivity to this combination. Pharmacodynamic responses and growth inhibitory effects of the combination were studied in xenografts of the same cell lines, as well as in primary human tumor xenografts, of RAS mutant melanoma and CRC. Results: MLN2480 inhibits MAPK pathway signaling in BRAF mutant and some RAS mutant preclinical cancer models at concentrations that are tolerated in vivo. MLN2480 is most potent in BRAF mutant melanoma models but also has single agent activity in some RAS mutant models. The combination of MLN2480 with TAK-733 inhibits the growth of a broader range of RAS mutant tumor models than single agent MLN2480, including primary human tumor xenograft models of melanoma and CRC. In vitro analysis of this drug combination in cell proliferation assays demonstrates synergistic activity. Western blot analysis demonstrated the effect of MLN2480 in reversing feedback activation of MEK in response to TAK-733, leading to more concerted MAPK pathway inhibition. Conclusions: The activity of the RAF kinase inhibitor MLN2480 in preclinical models of BRAF and RAS mutant melanoma and CRC provides a rationale for clinical testing. The combination of MLN2480 with the MEK inhibitor TAK-733 represents an additional strategy for clinical research within these tumor types. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C146. Citation Format: Elizabeth Grace Carideo Cunniff, Julie Zhang, Jouhara Chouitar, Jerome Mettetal, Kazuhide Nakamura, Takeo Arita, Akito Nakamura, Masanori Okaniwa, Tomoyasu Ishikawa, Sei Yoshida, Robyn Fabrey, Patrick Vincent, Kurt Eng, Khristofer Garcia, Deanna Borelli, Teena Vagrhese, Steve Stroud, Saurabh Menon, Mike Kuranda, Katherine Galvin. Combination treatment with the investigational RAF kinase inhibitor MLN2480 and the investigational MEK kinase inhibitor TAK-733 inhibits the growth of BRAF mutant and RAS mutant preclinical models of melanoma and CRC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C146.

Abstract C255: Discovery of TAK-632: A selective kinase inhibitor of pan-RAF with potent antitumor activity against BRAF and NRAS mutant melanomas.

Abstract The RAF family kinases play critical roles in cancer progression. Recently, BRAF selective inhibitors have shown significant clinical efficacy in melanoma patients bearing oncogenic BRAFV600E mutation. However, several studies reported that RAF inhibitors instinctively transactivate RAF homodimers (CRAF-CRAF) or heterodimers (CRAF-BRAF(wt)) and activate RAS dependent MAPK signaling. Along with this mechanism, it has been reported that selective BRAF inhibitors have not shown potent anti-proliferative activity against cancer cell lines such as NRAS mutant melanoma in which RAS dependent MAPK signaling is activated (Hong Yang et al., Cancer Res., 2010, 70, 5518-5527). However, our initial investigation using fibroblast CsFb (BRAFwt) cells indicated that phosphorylation of MEK and ERK was inhibited by some DFG-out inhibitors, but not by DFG-in inhibitors. These results led to the hypothesis: continuous inhibition of pan-RAF (BRAF and CRAF) with DFG-out type inhibitors could suppress the feedback activation. Here we report the discovery and characterization of pan-RAF inhibitor TAK-632. We designed novel 1,3-benzothiazole class derivatives using knowledge of structure-activity relationships gained from studies of our thiazolo[5,4-b]pyridine class RAF/VEGFR2 inhibitor (Masanori Okaniwa et al., J. Med. Chem., 2012, 55, 3452-3478). To enrich RAF kinase selectivity vs. VEGFR2, we utilized the cocrystal structures of our lead compound with both BRAF and VEGFR2. Eventually, we designed and selected 7-cyano derivative TAK-632 as a development candidate. Cocrystal structure analysis of BRAF bearing TAK-632 revealed that accommodation of the 7-cyano group into the BRAF-selectivity pocket and the 3-(trifluoromethyl)phenyl acetamide moiety into the hydrophobic back pocket of BRAF in the DFG-out conformation contributed to enhanced RAF inhibition and selectivity vs. VEGFR2. Reflecting its potent pan-RAF inhibition (IC50: BRAFV600E 2.4 nM, CRAF 1.4 nM) and slow dissociation (koff) profile measured by surface plasmon resonance (SPR) spectroscopy, TAK-632 demonstrated significant cellular activity against mutated BRAF or mutated NRAS cancer cell lines. Furthermore, in both A375 (BRAFV600E) and HMVII (NRASQ61K) xenograft models in rats, TAK-632 demonstrated regressive antitumor activity by twice daily, 14-day repetitive administration without significant body weight loss. In conclusion, these results raise the possibility of using slow off-rate pan-RAF inhibitors such as TAK-632 for the treatment of human cancers harboring either BRAFV600E or NRAS mutant. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C255. Citation Format: Masanori Okaniwa, Masaaki Hirose, Takeo Arita, Masato Yabuki, Akito Nakamura, Terufumi Takagi, Tomohiro Kawamoto, Noriko Uchiyama, Akihiko Sumita, Shunichirou Tsutsumi, Tsuneaki Tottori, Yoshitaka Inui, Bi-Ching Sang, Jason Yano, Kathleen Aertgeerts, Sei Yoshida, Tomoyasu Ishikawa. Discovery of TAK-632: A selective kinase inhibitor of pan-RAF with potent antitumor activity against BRAF and NRAS mutant melanomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C255.

Use of combination treatment with the investigational RAF kinase inhibitor MLN2480 and the investigational MEK kinase inhibitor TAK-733 on the growth of BRAF-mutant and RAS-mutant preclinical models of melanoma and CRC.

e13529 Background: RAS mutant melanoma and colorectal cancer represent areas of significant unmet medical need. MLN2480 is an investigational class II RAF kinase inhibitor and TAK-733 is an investigational allosteric MEK kinase inhibitor; each of which is the subject of a single agent phase I clinical trial. The present studies have characterized the combination activity of these agents in BRAF mutant and RAS mutant preclinical models of melanoma and colorectal cancer. Methods: Combination effects of MLN2480 and TAK-733 on cell viability were studied using an ATP-based cell viability assay across a panel of BRAF and RAS mutant melanoma and CRC cell lines. Western blot analysis was used to compare effects on MAPK pathway signaling and response markers in cell lines showing a range of sensitivity to this combination. Pharmacodynamic responses and growth inhibitory effects of the combination were studied in xenografts of the same cell lines, as well as in primary human tumor xenografts, of RAS mutant melanoma and CRC. Results: MLN2480 inhibits MAPK pathway signaling in BRAF mutant and some RAS mutant preclinical cancer models at concentrations that are tolerated in vivo. MLN2480 is most potent in BRAF mutant melanoma models but also has single agent activity in some RAS mutant models. The combination of MLN2480 with TAK-733 inhibits the growth of a broader range of RAS mutant tumor models than single agent MLN2480, including primary human tumor xenograft models of melanoma and CRC. In vitroanalysis of this drug combination in cell proliferation assays demonstrates synergistic activity. Western blot analysis demonstrated the effect of MLN2480 in reversing feedback activation of MEK in response to TAK-733, leading to more concerted MAPK pathway inhibition. Conclusions: The activity of the RAF kinase inhibitor MLN2480 in preclinical models of BRAF and RAS mutant melanoma and CRC provides a rationale for clinical testing. The combination of MLN2480 with the MEK inhibitor TAK-733 represents an additional strategy for clinical research within these tumor types.

Antitumor effects of the investigational selective MEK inhibitor TAK733 against cutaneous and uveal melanoma cell lines

BackgroundTAK733 is a novel allosteric, non-ATP-binding, inhibitor of the BRAF substrates MEK-1/2.MethodsThe growth inhibitory effects of TAK733 were assessed in a panel of 27 cutaneous and five uveal melanoma cell lines genotyped for driver oncogenic mutations. Flow cytometry, Western blots and metabolic tracer uptake assays were used to characterize the changes induced by exposure to TAK733.ResultsFourteen cutaneous melanoma cell lines with different driver mutations were sensitive to the antiproliferative effects of TAK733, with a higher proportion of BRAFV600E mutant cell lines being highly sensitive with IC50s below 1 nM. The five uveal melanoma cell lines had GNAQ or GNA11 mutations and were either moderately or highly sensitive to TAK733. The tested cell lines wild type for NRAS, BRAF, GNAQ and GNA11 driver mutations were moderately to highly resistant to TAK733. TAK733 led to a decrease in pERK and G1 arrest in most of these melanoma cell lines regardless of their origin, driver oncogenic mutations and in vitro sensitivity to TAK733. MEK inhibition resulted in increase in pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. Uptake of the metabolic tracers FDG and FLT was inhibited by TAK733 in a manner that closely paralleled the in vitro sensitivity assays.ConclusionsThe MEK inhibitor TAK733 has antitumor properties in melanoma cell lines with different oncogenic mutations and these effects could be detectable by differential metabolic tracer uptake.

Abstract 3738: A rationale for combining the targeted investigational agents TAK-733, a MEK1/2 inhibitor, with alisertib (MLN8237), an Aurora A kinase inhibitor, for cancer therapy

Abstract MEK1/2 kinases have diverse functions in cell proliferation and survival, including the regulation of cell cycle transitions between the G1, S, and G2 phases and mediation of DNA damage response pathways. Therefore, inhibition of MEK1/2 may facilitate the antiproliferative effects of other therapies that disrupt cell cycle progression leading to DNA damage, including antimitotic therapies. AAK is a serine/threonine protein kinase essential for normal mitotic progression. Inhibition of AAK leads to inappropriate progression through mitosis in the presence of misaligned chromosomes resulting in aneuploidy and DNA damage, which leads to apoptosis or senescence. Here, we evaluated the combination of the allosteric MEK1/2 inhibitor TAK-733 with alisertib, the selective AAK inhibitor. Proliferation assays in multiple tumor cell lines showed a concentration-dependent decrease in the IC50 of TAK-733 and alisertib with increasing concentrations of the second agent, suggesting increased anti-proliferative activity of these agents when combined. This result was corroborated by measuring changes in cell confluence over time, a surrogate for proliferation rate, using an IncuCyte live-cell imaging system. The decreased proliferation rate of the combination was due in part to induction of apoptosis, as demonstrated by Annexin V and sub-diploid DNA content analysis. Cell cycle progression differences of the TAK-733/alisertib combination relative to the single agents were assessed using time-lapse video microscopy by measuring the time to the first mitotic event after drug addition, time spent in mitosis, and time between mitotic events. Of these, the only observed difference in cell-cycle progression time in the A2780 ovarian carcinoma cell line was the time between mitotic events, which was 10 hours longer for the combination relative to single agent alisertib. Moreover, the combination of TAK-733 and alisertib significantly reduced the ability of the cells to re-enter the cell cycle after one mitosis compared to either single agent; 30% for the combination, compared to 90% for TAK-733 and 76% for alisertib. Flow cytometry DNA content analysis showed that TAK-733 suppressed the formation of &amp;gt;4N/tetraploid cells induced by alisertib alone in multiple cell lines. This observed decrease in the proportion of &amp;gt;4N cells is greater than the decrease that could be extrapolated solely from the cell cycle-slowing effect of the combination, suggesting that TAK-733 reduces the proliferative potential of the alisertib-generated aneuploid cells via cell-cycle arrest and apoptosis. Collectively, these findings demonstrate an added benefit of TAK-733 over alisertib alone and provide a pre-clinical rationale for further investigation of the combination of TAK-733 and alisertib for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3738. doi:1538-7445.AM2012-3738

Abstract B136: Application of a mathematical model to understand the cell cycle progression effects of MEK1/2 kinase inhibition using the investigational small-molecule allosteric inhibitor TAK-733.

Abstract In order to more precisely understand the effects of MEK1/2 inhibition on cell cycle progression, cellular imaging and biochemical assays were coupled with mathematical modeling. The MEK/ERK signaling cascade modulates multiple outcomes relevant to cancer therapy including cell growth, proliferation, differentiation and apoptosis. Moreover, MEK/ERK signaling regulates transitions between the G1, S and G2 cell cycle phases, and mediates the response to DNA damage induced by a wide variety of DNA damaging agents. Inhibition of the MEK/ERK pathway represents a promising approach for the treatment of cancer. A number of small-molecule inhibitors are currently in clinical trials, including TAK-733, a novel, investigational, selective, non ATP competitive, allosteric inhibitor of MAP kinase-ERK kinase (MEK) activity. MEK inhibition is often described to invoke a complete arrest at the G1-S cell cycle transition leading to a rapid cessation of cell growth. In order to assess this, the kinetics of cell cycle progression using continuous quantitative techniques in several different cell types was examined. Time-lapse microscopy studies and confluence analyses using an IncuCyte system demonstrated that cells treated with TAK-733 continue to divide over a period of five days, albeit at a slower rate than the DMSO treated cells. These observations were corroborated with flow cytometry using CFSE, a fluorescent cytoplasmic dye whose rate of decay is a measure of the cell division rate, and with immunofluorescence microscopy using the G2/M marker cyclin B. This continued cell cycling in the presence of TAK-733 was demonstrated to occur with abnormal cell cycle progression times and abnormal DNA content profiles, suggesting altered rates of passage through S and G2/M phase. A mathematical model was constructed to quantitatively describe how MEK inhibition is affecting the passage of the cell population through the cell cycle. When the model is fitted to the experimentally observed data, it inferred the rate of entrance and exit from each phase of the cell cycle under both control and treatment conditions. The model simultaneously captured both the observed accumulation in G0/G1 as well as the observed progression through the cell cycle during treatment. Interestingly, it was able to explain both of these results without invoking a complete G1 arrest. Instead it showed that the rate of exit from G1 is reduced upon inhibition. These findings were supported by time-lapse microscopy observations which were not used in fitting the model. This reassessment of the canonical view of cell-cycle progression defects following MEK1/2 inhibition by means of the mathematical modeling approach described here may provide key differentiation opportunities in the rational development of TAK-733 and other MEK1/2 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B136.

Abstract C182: Antitumor efficacy of the investigational MEK inhibitor TAK-733 against melanoma cell lines and human-derived tumor explant models.

Abstract Background: Sixty-percent of melanomas express mutant BRAF oncoproteins, which result in constitutive activation of the Rasmitogen activated protein kinase (MAPK) pathway and thus abnormal growth and ultimately a malignant phenotype. It has also been demonstrated that many melanomas are dependent on the MAPK pathway for survival and proliferation. MEK is a seronine/threonine kinase that is part of the BRAF downstream signaling cascade and is responsible for the phosphorylation of ERK1/2. Interestingly, ERK1/2 appear to be the sole target of the MEK protein kinase. Without ERK1/2 nuclear translocation, the inappropriate nuclear signaling responsible for the malignant phenotype is inhibited. The development of BRAF inhibitors has provided an exciting new treatment option for BRAF mutated melanoma, but unfortunately many patients have relapsed due to downstream alternative pathway activation. Investigational drug TAK-733 (Millennium Pharmaceuticals, Inc) is a potent, novel, selective, non-ATP competitive, allosteric inhibitor of MEK 1/2. With molecular inhibition downstream of BRAF, CRAF, COT, or NRAS mutations, tumor growth would theoretically be slowed or suppressed altogether. We explored the preclinical efficacy of TAK-733 in both in vitro and in vivo models of melanoma. Based on these translational results, TAK-733 demonstrates promising pre-clinical data supportive of further clinical development. Methods &amp; Results: 36 melanoma cell lines were exposed to TAK-733 and the IC50 was determined using the SRB method. The cell lines were then classified as sensitive (S) or resistant (R) based on an IC50&amp;lt;0.01uM or IC50&amp;gt;0.1uM, respectively. BRAF status was obtained on all cell lines and did not correlate with responsiveness to TAK-733. Immunoblotting for effector proteins was performed on both S and R cell lines after 72 hrs of treatment with TAK-733. Phospho-ERK was suppressed in both S and R cell lines, confirming TAK-733-mediated inhibition of MEK 1/2. The evidence of MEK inhibition in the relatively resistant cell lines suggests other escape pathways are also contributing to melanoma survival and proliferation. There was no evidence of up-regulation of known MAPK-associated alternative pathways involving PI3 kinase or phospho-S6. Ten patient-derived melanoma tumors (5 V600E BRAF mutant, 2 wild-type BRAF, 3 unknown) were implanted into nude mice and treated daily via oral gavage with TAK-733 10mg/kg or 25mg/kg daily. The tumors were measured three times weekly and tumor growth inhibition (TGI) was analyzed. Nine explants, regardless of BRAF status, showed statistically significant TGI, ranging 87–97%, when compared to controls. One of the ten explants (BRAF V600E mutant) was resistant to TAK-733 with a TGI of 23%. Additionally, in one sensitive explant (BRAF wild type), treatment with TAK-733 was stopped, re-growth to 1533 mm3 was allowed, and subsequent re-treatment with TAK-733 induced tumor shrinkage to &amp;lt;50mm3. The compound was well-tolerated and no significant weight loss or lethality was observed. Conclusion: These data demonstrate that TAK-733 exhibited robust tumor growth inhibition and regression in human melanoma cell lines and human melanoma explant models in mice. Based on these results, TAK-733 demonstrates promising preclinical data supportive of further clinical development in melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C182.

Abstract 3584: Effects of the investigational selective MEK inhibitor TAK-733 against cutaneous and uveal melanoma cell lines

Abstract Background. There are several MEK inhibitors currently under clinical investigation, including, TAK-733, a novel non-ATP-competitive allosteric selective inhibitor of MEK. We have analyzed the sensitivity of human melanoma cell lines of different origins to TAK-733. Methods. The half maximal inhibitory concentration (IC50) of TAK-733 was assessed in a panel of 27 skin melanoma cell lines and 5 ocular melanoma cell lines genotyped for driver and secondary oncogenic mutations. Flow cytometry was used to analyze effects on cell cycle progression, and Western blots to analyze the downstream signaling changes upon exposure to TAK-733. Results. Among 12 BRAFV600E mutated cell lines tested, 6 were sensitive to TAK-733 with IC50s less than 10 nM, including 4 highly sensitive cell lines with IC50s below 1 nM. In 5 cell lines with BRAFV600E mutation the IC50 was higher than 100 nM and were considered highly resistant. Among 10 NRASQ61L mutant melanoma cell lines, 6 were sensitive with IC50s below 10 nM. Among 5 melanoma cell lines with wild type sequences for NRAS, BRAF, GNAQ and GNA11 genes only 1 was sensitive to TAK-733. All 5 of the uveal melanoma cell lines were highly sensitive to TAK-733 IC50 regardless of having a GNAQ or GNA11 mutations. MEK inhibition resulted in increase of pMEK more prominently in NRASQ61L mutant and GNAQ mutant cell lines than in BRAFV600E mutant cell lines. In all cases TAK-733 induced marked dose-dependent inhibition of pERK, regardless of the driver oncogenic mutation or the sensitivity or resistance to this agent in cell viability assays. Cell lines resistant to TAK-733 showed no inhibition of pAKT or pS6K, while there was a general trend towards inhibition of these two phosphorylated molecules in sensitive cell lines. Conclusions. TAK-733 is a potent MEK inhibitor with high antitumor activity against human melanoma cell lines with BRAF, NRAS, GNAQ and GNA11 driver mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3584. doi:10.1158/1538-7445.AM2011-3584

1449 ORAL A phase I/II trial to assess tolerability and efficacy of RAD-001 with gefitinib in patients with glioblastoma multiforme and castrate metastatic prostate cancer

Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. Primary DIPG tumors harbor a number of mutations including alterations in PTEN, AKT, and PI3K and exhibit activation of mammalian Target of Rapamycin Complex 1 and 2 (mTORC1/2). mTORC1/2 regulate protein translation, cell growth, survival, invasion, and metabolism. Pharmacological inhibition of mTORC1 is minimally effective in DIPG. However, the activity of dual TORC kinase inhibitors has not been examined in this tumor type.Nanomolar levels of the mTORC1/2 inhibitor TAK228 reduced expression of p-AKTS473 and p-S6S240/244 and suppressed the growth of DIPG lines JHH-DIPG1, SF7761, and SU-DIPG-XIII. TAK228 induced apoptosis in DIPG cells and cooperated with radiation to further block proliferation and enhance apoptosis.TAK228 monotherapy inhibited the tumorigenicity of a murine orthotopic model of DIPG, more than doubling median survival (p = 0.0017) versus vehicle. We conclude that dual mTOR inhibition is a promising potential candidate for DIPG treatment.