Encorafenib, a selective BRAF inhibitor (BRAFi), has a pharmacologic profile that is distinct from that of other clinically active BRAFis. We evaluated encorafenib in a phase I study in patients with ...BRAFi treatment-naïve and pretreated
-mutant melanoma.
The pharmacologic activity of encorafenib was first characterized preclinically. Encorafenib monotherapy was then tested across a range of once-daily (50-700 mg) or twice-daily (75-150 mg) regimens in a phase I, open-label, dose-escalation and -expansion study in adult patients with histologically confirmed advanced/metastatic
-mutant melanoma. Study objectives were to determine the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D), characterize the safety and tolerability and pharmacokinetic profile, and assess the preliminary antitumor activity of encorafenib.
Preclinical data demonstrated that encorafenib inhibited BRAF V600E kinase activity with a prolonged off-rate and suppressed proliferation and tumor growth of
V600E-mutant melanoma models. In the dose-escalation phase, 54 patients (29 BRAFi-pretreated and 25 BRAFi-naïve) were enrolled. Seven patients in the dose-determining set experienced dose-limiting toxicities. Encorafenib at a dose of 300 mg once daily was declared the RP2D. In the expansion phase, the most common all-cause adverse events were nausea (66%), myalgia (63%), and palmar-plantar erythrodysesthesia (54%). In BRAFi-naïve patients, the overall response rate (ORR) and median progression-free survival (mPFS) were 60% and 12.4 months 95% confidence interval (CI), 7.4-not reached (NR). In BRAFi-pretreated patients, the ORR and mPFS were 22% and 1.9 months (95% CI, 0.9-3.7).
Once-daily dosing of single-agent encorafenib had a distinct tolerability profile and showed varying antitumor activity across BRAFi-pretreated and BRAFi-naïve patients with advanced/metastatic melanoma.
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Anaplastic lymphoma kinase (
) is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an
screen for synergistic drug combinations that target neuroblastomas with ...mutations in
to determine whether drug combinations could enhance antitumor efficacy.
We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines. We investigated the combination of ceritinib and ribociclib on
proliferation, cell cycle, viability, caspase activation, and the cyclin D/CDK4/CDK6/RB and pALK signaling networks in cell lines with representative ALK status. We performed
trials in CB17 SCID mice bearing conventional and patient-derived xenograft models comparing ceritinib alone, ribociclib alone, and the combination, with plasma pharmacokinetics to evaluate for drug-drug interactions.
The combination of ribociclib, a dual inhibitor of cyclin-dependent kinase (CDK) 4 and 6, and the ALK inhibitor ceritinib demonstrated higher cytotoxicity (
= 0.008) and synergy scores (
= 0.006) in cell lines with
mutations as compared with cell lines lacking mutations or alterations in
Compared with either drug alone, combination therapy enhanced growth inhibition, cell-cycle arrest, and caspase-independent cell death. Combination therapy achieved complete regressions in neuroblastoma xenografts with
-F1174L and F1245C
resistance mutations and prevented the emergence of resistance. Murine ribociclib and ceritinib plasma concentrations were unaltered by combination therapy.
This preclinical combination drug screen with
validation has provided the rationale for a first-in-children trial of combination ceritinib and ribociclib in a molecularly selected pediatric population.
.
Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to ...hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition.
We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies.
Sensitivity to binimetinib and ribociclib was inversely related (
= -0.58,
= 0.009).
amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs.
Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling.
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Resistance to the RAF inhibitor vemurafenib arises commonly in melanomas driven by the activated BRAF oncogene. Here, we report antitumor properties of RAF709, a novel ATP-competitive kinase ...inhibitor with high potency and selectivity against RAF kinases. RAF709 exhibited a mode of RAF inhibition distinct from RAF monomer inhibitors such as vemurafenib, showing equal activity against both RAF monomers and dimers. As a result, RAF709 inhibited MAPK signaling activity in tumor models harboring either BRAF
alterations or mutant N- and KRAS-driven signaling, with minimal paradoxical activation of wild-type RAF. In cell lines and murine xenograft models, RAF709 demonstrated selective antitumor activity in tumor cells harboring BRAF or RAS mutations compared with cells with wild-type BRAF and RAS genes. RAF709 demonstrated a direct pharmacokinetic/pharmacodynamic relationship in
tumor models harboring KRAS mutation. Furthermore, RAF709 elicited regression of primary human tumor-derived xenograft models with BRAF, NRAS, or KRAS mutations with excellent tolerability. Our results support further development of inhibitors like RAF709, which represents a next-generation RAF inhibitor with unique biochemical and cellular properties that enables antitumor activities in RAS-mutant tumors.
In an effort to develop RAF inhibitors with the appropriate pharmacological properties to treat RAS mutant tumors, RAF709, a compound with potency, selectivity, and
properties, was developed that will allow preclinical therapeutic hypothesis testing, but also provide an excellent probe to further unravel the complexities of RAF kinase signaling.
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The use of RNA interference (RNAi) has enabled loss-of-function studies in mammalian cancer cells and has hence become critical for identifying and validating cancer drug targets. Current transient ...siRNA and stable shRNA systems, however, have limited utility in accurately assessing the cancer dependency due to their short-lived effects and limited in vivo utility, respectively. In this study, a single-vector lentiviral, Tet-inducible shRNA system (pLKO-Tet-On) was generated to allow for the rapid generation of multiple stable cell lines with regulatable shRNA expression. We demonstrate the advantages and versatility of this system by targeting two polycomb group proteins, Bmi-1 and Mel-18, in a number of cancer cell lines. Our data show that pLKO-Tet-On-mediated knockdown is tightly regulated by the inducer tetracycline and its derivative, doxycycline, in a concentration- and time-dependent manner. Furthermore, target gene expression is fully restored upon withdrawal of the inducing agent. An additional, 17 distinct gene products have been targeted by inducible shRNAs with robust regulation in all cases. Importantly, we functionally validate the ability of the pLKO-Tet-On vector to reversibly silence targeted transcripts in vivo. The versatile and robust inducible lentiviral RNAi system reported herein can therefore serve as a powerful tool to rapidly reveal tumor cell dependence.
Epigenetic dysregulation is an emerging hallmark of cancers. We developed a high-information-content mass spectrometry approach to profile global histone modifications in human cancers. When applied ...to 115 lines from the Cancer Cell Line Encyclopedia, this approach identified distinct molecular chromatin signatures. One signature was characterized by increased histone 3 lysine 36 (H3K36) dimethylation, exhibited by several lines harboring translocations in NSD2, which encodes a methyltransferase. A previously unknown NSD2 p.Glu1099Lys (p.E1099K) variant was identified in nontranslocated acute lymphoblastic leukemia (ALL) cell lines sharing this signature. Ectopic expression of the variant induced a chromatin signature characteristic of NSD2 hyperactivation and promoted transformation. NSD2 knockdown selectively inhibited the proliferation of NSD2-mutant lines and impaired the in vivo growth of an NSD2-mutant ALL xenograft. Sequencing analysis of >1,000 pediatric cancer genomes identified the NSD2 p.E1099K alteration in 14% of t(12;21) ETV6-RUNX1-containing ALLs. These findings identify NSD2 as a potential therapeutic target for pediatric ALL and provide a general framework for the functional annotation of cancer epigenomes.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Alterations in
occur in cancers, both in the treatment-naïve state and following targeted therapies, most notably BRAF and MEK inhibitors in
-V600E-mutant melanoma and colorectal cancer. Efforts were ...undertaken to understand the effects of these mutations, based upon protein structural location, and MEK1/2 activity. Two categories of MEK1/2 alterations were evaluated, those associated with either the allosteric pocket or helix-A. Clinically, MEK1/2 alterations of the allosteric pocket are rare and we demonstrate that they confer resistance to MEK inhibitors, while retaining sensitivity to BRAF inhibition. Most mutations described in patients fall within, or are associated with, helix-A. Mutations in this region reduce sensitivity to both BRAF and MEK inhibition and display elevated phospho-ERK1/2 levels, independent from increases in phospho-MEK1/2. Biochemical experiments with a representative helix-A variant,
-Q56P, reveal both increased catalytic efficiency of the activated enzyme, and phosphorylation-independent activity relative to wild-type MEK1. Consistent with these findings, MEK1/2 alterations in helix A retain sensitivity to downstream antagonism via pharmacologic inhibition of ERK1/2. This work highlights the importance of classifying mutations based on structural and phenotypic consequences, both in terms of pathway signaling output and response to pharmacologic inhibition.
This study suggests that alternate modes of target inhibition, such as ERK inhibition, will be required to effectively treat tumors harboring these MEK1/2-resistant alleles.
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Reoccurring/high-risk neuroblastoma (NB) tumors have the enrichment of non-RAS/RAF mutations along the mitogen-activated protein kinase (MAPK) signaling pathway, suggesting that activation of MEK/ERK ...is critical for their survival. However, based on preclinical data, MEK inhibitors are unlikely to be active in NB and have demonstrated dose-limiting toxicities that limit their use. Here, we explore an alternative way to target the MAPK pathway in high-risk NB. We find that NB models are among the most sensitive among over 900 tumor-derived cell lines to the allosteric SHP2 inhibitor SHP099. Sensitivity to SHP099 in NB is greater in models with loss or low expression of the RAS GTPase activation protein (GAP) neurofibromin 1 (NF1). Furthermore, NF1 is lower in advanced and relapsed NB and NF1 loss is enriched in high-risk NB tumors regardless of MYCN status. SHP2 inhibition consistently blocks tumor growth in high-risk NB mouse models, revealing a new drug target in relapsed NB.
Display omitted
•We identify neuroblastoma models as sensitive to SHP2 inhibition•SHP099 sensitivity is correlated with low expression of neurofibromin (NF1)•SHP2 inhibitors inhibit pERK in neuroblastoma cells better than in normal cells•NF1 mutation/expression may predict SHP2 inhibitor response in neuroblastoma
In this paper, Cai et al. demonstrate that high-risk neuroblastomas with low NF1 expression are sensitive to SHP2 inhibitors, which may have treatment advantages over MEK inhibitors. Targeting SHP2 blocks neuroblastoma tumor growth. As several SHP2 inhibitors are in clinical trials, SHP2 inhibitors may benefit high-risk NB patients.
Abstract
Background: The cyclin-dependent kinase (CDK) 4/6 inhibitors ribociclib, abemaciclib, and palbociclib have been established as effective treatment options for hormone receptor–positive (HR+) ...human epidermal growth factor receptor 2–negative (HER2–) breast cancer. While these agents have not been evaluated head-to-head in clinical studies, they have all demonstrated significant progression-free survival improvements; however, only two of these, abemaciclib plus endocrine therapy (ET; MONARCH 2) and ribociclib plus ET (MONALEESA-3 and MONALEESA-7), achieved significant overall survival (OS) to date prompting closer examination of how these CDK4/6 inhibitors are distinct. Preclinical studies revealed differences in how these molecules interact with various kinases. These studies indicated that ribociclib and palbociclib exhibited greater selectivity for CDK4 and CDK6 relative to other human kinases than abemaciclib. In addition, studies suggested that these molecules displayed different relative activity against their primary targets CDK4 and CDK6. Although intriguing, these latter studies either used purified proteins without the accompanying cellular context or used proliferation as a proxy for target engagement. Here, we sought to extend the body of evidence created by the prior analyses by constructing cellular model systems where the effects of each CDK4/6 inhibitor on CDK4 or CDK6 could be studied in isolation.Methods: MEL-JUSO (an NRAS mutant melanoma cell line) and MIA PaCa-2 (a KRAS mutant pancreatic ductal adenocarcinoma cell line) cells were selected for this analysis after being identified as lacking dependence on either CDK4 or CDK6, as indicated by short hairpin RNA knockdown. Isogenic variants of each cell line lacking either CDK4 or CDK6 expression were generated by ablating CDK4 or CDK6 expression using CRISPR/CAS9 genome editing techniques and single-cell clonal selection. Levels of phosphorylated RB (phospho-RB) protein were used as a readout for target inhibition and were measured by Fast Scan phospho-RB (ppRB807/811) enzyme-linked immunosorbent assay (ELISA). Half-maximal inhibitory concentrations (IC50) for each CDK4/6 inhibitor were calculated in parental cells, as well as variant cell lines expressing only CDK4 or CDK6. Additionally, measurement of phospho-RB was performed in T47-D cells (an estrogen receptor–positive breast cancer cell line) to confirm results of prior analyses.Results: The level of phospho-RB inhibition in T47-D cells was similar to what has been described previously (Chen P, et al. Mol Cancer Ther. 2016). In MEL-JUSO cells, ribociclib, abemaciclib, and palbociclib inhibited CDK4 at 11-, 22-, and 2-fold lower drug concentrations than CDK6, respectively. In MIA PaCa-2 cells, ribociclib, abemaciclib, and palbociclib inhibited CDK4 at 9-, >47-, and 2-fold lower drug concentrations than CDK6, respectively. Conclusions: Consistent with prior biochemical studies and cell proliferation assays, our findings indicate that both ribociclib and abemaciclib more potently inhibit CDK4 than CDK6, whereas palbociclib has similar activity against both targets in cells. Understanding the importance of CDK4 relative to CDK6 in the etiology of breast cancer and possible implications for increased CDK4 target engagement is an emerging topic of discussion in the field. Given that CDK4 has been shown to be expressed at higher levels in breast tumor samples, and many breast cancer cell lines have demonstrated greater dependence on CDK4 vs CDK6, the differential inhibition of the CDK4/6 inhibitors may have important implications.
Citation Format: Scott Delach, Giordano Caponigro. Preclinical head-to-head comparison of CDK4/6 inhibitor activity toward CDK4 vs CDK6 abstract. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-10.
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