RAS Mutant Models Research Articles

Separable cell cycle arrest and immune response elicited through pharmacological CDK4/6 and MEK inhibition in RASmut disease models.

The combination of CDK4/6 and MEK inhibition as a therapeutic strategy has shown promise in various cancer models, particularly in those harboring RAS mutations. An initial high-throughput drug screen identified a high synergy between the CDK4/6 inhibitor palbociclib and the MEK inhibitor trametinib when used in combination in soft tissue sarcomas. In RAS mutant models, combination treatment with palbociclib and trametinib induced significant G1 cell cycle arrest, resulting in a marked reduction in cell proliferation and growth. CRISPR-mediated RB1 depletion resulted in a decreased response to CDK4/6 and MEK inhibition, which was validated in both cell culture and xenograft models. Beyond its cell cycle inhibitory effects, pathway enrichment analysis revealed the robust activation of interferon pathways upon CDK4/6 and MEK inhibition. This induction of gene expression was associated with the upregulation of retroviral elements. The TBK1(TANK-binding kinase 1) inhibitor GSK8612 selectively blocked the induction of interferon-related genes induced by palbociclib and trametinib treatment, and highlighted the separable epigenetic responses elicited by combined CDK4/6 and MEK inhibition. Together, these findings provide key mechanistic insights into the therapeutic potential of CDK4/6 and MEK inhibition in soft tissue sarcoma.

Abstract LB-114: RAF inhibitor LXH254 effectively inhibits B-and-CRAF, but not ARAF

Abstract RAS mutant tumors have proven largely refractory to inhibitors of the MAPK pathway in clinic. In contrast, selective inhibitors of BRAFV600E but not wild type RAF have been successfully developed in BRAFV600E mutant melanoma, NSCLC, and thyroid cancers. Success of these drugs results largely from the therapeutic index imparted by lack of activity against wild RAFs; however, this selectivity also prevents their utility in indications beyond BRAFV600E disease, such as RAS-mutant tumors. In contrast, MEK1/2 inhibitors, while effective pre-clinically in RAS mutant models, have proven ineffective against RAS mutant tumors due at least in part to the poor therapeutic index of these molecules that results from pathway inhibition in normal cells. Thus, to inhibit effectively MAPK pathway signaling in tumors, inhibitors with improved tolerability via differential pathway inhibition in tumor vs normal tissues are required. Recent data in mice conditionally lacking different RAF paralogs suggest that inhibitors biased towards CRAF might retain anti-tumor properties, with improved tolerability. LXH254 is a RAF inhibitor with high selectivity and nanomolar potency for BRAF and CRAF. Profiling of LXH254 activity in Ras mutant lines lacking either ARAF, BRAF or CRAF by CRISPR-Cas9 modification revealed that loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. This increased sensitivity to LXH254 from ARAF knockout was rescued by over-expression of wild-type ARAF but not by either ARAF that harbored mutations that impaired kinase activity (K336M, D447A) or its ability to dimerize (R362H). Comparing the effect of LXH254 on MAPK pathway signaling in RAS mutant cells engineered to express only one of the three RAF paralogs indicated that significantly higher concentrations of LXH254 are required to inhibit MAPK signaling in cells expressing only ARAF. Furthermore, at low concentrations of LXH254, cells expressing only ARAF displayed paradoxical activation of MAPK signaling in a manner similar to the BRAFV600E selective inhibitor dabrafenib. Thus, both LXH254 and dabrafenib inhibit mutationally activated monomeric BRAF, and promote/stabilize RAS-dependent dimerization of WT RAFs. However, because dabrafenib is only able to effectively inhibit one of the two RAF protomers in each dimer it paradoxically activates MAPK signaling in a RAS-dependent manner. In contrast, LXH254 retains activity against dimerized B-and C-RAF (but not ARAF) thereby theoretically extending its utility beyond BRAF mutant disease with better tolerability relative to, e.g. MEK1/2 inhibitors. Citation Format: Kelli-Ann Monaco, Scott Delach, Paul Fordjour, Salonee Parikh, Yun Feng, Mariela Jaskelioff, Darrin Stuart, Giordano Caponigro. RAF inhibitor LXH254 effectively inhibits B-and-CRAF, but not ARAF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-114.

Correction: Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

[This corrects the article DOI: 10.1371/journal.pone.0185862.].

Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.

Abstract 187: ASN0007, a potent ERK1/2 inhibitor with strong antitumor activity in multiple RAS mutant models

Abstract The extracellular-signal-regulated kinases, ERK1 and ERK2 (ERK1/2), play a critical role in the RAS/RAF/MEK signaling pathway which is quite frequently activated by mutations in upstream targets such as BRAF, RAS and receptor tyrosine kinases. Most of the resistance mechanisms to BRAF and MEK inhibitors ultimately lead to increase in phosphorylation of ERK1/2 suggesting the importance of this node in the RAS/RAF/MEK pathway even in the resistance setting. Therefore, inhibition of ERK1/2 offers a promising strategy to address both innate and acquired resistance to BRAF and MEK inhibitors in various solid tumors. In order to inhibit this critical node of the RAS/RAF/MEK pathway, we discovered and characterized ASN007, a novel compound that shows potent inhibition of ERK1/2 kinases in a biochemical assay with low single-digit IC50 values. In mechanistic cell-based assays, ASN007 inhibited the phosphorylation of ERK1/2 substrates such as pRSK1, FRA1 and Elk1 in various cell lines. As expected, the compound did not have any effect on the phosphorylation of ERK1/2. ASN007 showed strong anti-proliferative activity in both BRAF and RAS mutant cell lines. Importantly, when compared to other ERK1/2 inhibitors such as BVD-523 and GDC-0994, ASN007 showed potent antiproliferative activity in a much broader panel of KRAS, NRAS and HRAS mutant cell lines representing various histological tumor types. The compound also caused cell cycle arrest at the G0-G1 phase and induced apoptosis in both BRAF and RAS mutant cell lines. In a daily oral dosing regimen, ASN007 demonstrated strong inhibition of tumor growth in multiple BRAF and KRAS mutant xenograft models in mice and was well tolerated at efficacious doses. ASN007 has a good pharmacokinetic profile in pre-clinical species and does not exhibit significant inhibition of CYP enzymes or hERG channel. Based on its profile in preclinical studies, ASN007 is expected to show strong efficacy in BRAF and RAS mutant cancers. ASN007 is currently in preclinical development. Citation Format: Sanjeeva P. Reddy, Dhanalakshmi Sivanandhan, Raghava Reddy Kethiri, Sreekala Nair, Purushottam Dewang, Mohd Zainuddin, Krishnakumar Vaithilingam, Roger A. Smith, Sandeep Gupta, Scott K. Thompson. ASN0007, a potent ERK1/2 inhibitor with strong antitumor activity in multiple RAS mutant models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 187.

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.

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.