Over the past two decades there has been a great deal of interest in the development of inhibitors of the cyclin-dependent kinases (CDKs). This attention initially stemmed from observations that ...different CDK isoforms have key roles in cancer cell proliferation through loss of regulation of the cell cycle, a hallmark feature of cancer. CDKs have now been shown to regulate other processes, particularly various aspects of transcription. The early non-selective CDK inhibitors exhibited considerable toxicity and proved to be insufficiently active in most cancers. The lack of patient selection biomarkers and an absence of understanding of the inhibitory profile required for efficacy hampered the development of these inhibitors. However, the advent of potent isoform-selective inhibitors with accompanying biomarkers has re-ignited interest. Palbociclib, a selective CDK4/6 inhibitor, is now approved for the treatment of ER+/HER2- advanced breast cancer. Current developments in the field include the identification of potent and selective inhibitors of the transcriptional CDKs; these include tool compounds that have allowed exploration of individual CDKs as cancer targets and the determination of their potential therapeutic windows. Biomarkers that allow the selection of patients likely to respond are now being discovered. Drug resistance has emerged as a major hurdle in the clinic for most protein kinase inhibitors and resistance mechanism are beginning to be identified for CDK inhibitors. This suggests that the selective inhibitors may be best used combined with standard of care or other molecularly targeted agents now in development rather than in isolation as monotherapies.
RAF inhibitors such as vemurafenib and dabrafenib block BRAF-mediated cell proliferation and achieve meaningful clinical benefit in the vast majority of patients with BRAF(V600E)-mutant melanoma. ...However, some patients do not respond to this regimen, and nearly all progress to therapeutic resistance. We used a pooled RNA interference screen targeting more than 16,500 genes to discover loss-of-function events that could drive resistance to RAF inhibition. The highest ranking gene was NF1, which encodes neurofibromin, a tumor suppressor that inhibits RAS activity. NF1 loss mediates resistance to RAF and mitogen-activated protein kinase (MAPK) kinase kinase (MEK) inhibitors through sustained MAPK pathway activation. However, cells lacking NF1 retained sensitivity to the irreversible RAF inhibitor AZ628 and an ERK inhibitor. NF1 mutations were observed in BRAF-mutant tumor cells that are intrinsically resistant to RAF inhibition and in melanoma tumors obtained from patients exhibiting resistance to vemurafenib, thus showing the clinical potential for NF1-driven resistance to RAF/MEK-targeted therapies.
RAF and MEK inhibitors are effective in BRAF-mutant melanoma but not in BRAF-mutant colorectal cancer. To gain additional insights into this difference, we performed a genome-scale pooled shRNA ...enhancer screen in a BRAF-mutant, RAF inhibitor-resistant colorectal cancer cell line exposed to the selective RAF inhibitor PLX4720. We identified multiple genes along the receptor tyrosine kinase (RTK)/mitogen-activated protein kinase (MAPK) signaling axis that, when suppressed, either genetically or pharmacologically, sensitized cells to the selective RAF inhibitor through sustained inhibition of MAPK signaling. Strikingly, CRAF was a key mediator of resistance that could be overcome by the use of pan-RAF inhibitors in combination with a MEK inhibitor. Furthermore, the combination of pan-RAF and MEK inhibitors displayed strong synergy in melanoma and colorectal cancer cell lines with RAS-activating events such as RTK activation, KRAS mutation, or NF1 loss-of-function mutations. Combinations of selective RAF inhibitors, such as PLX4720 or dabrafenib, with MEK inhibitors did not incur such profound synergy, suggesting that inhibition of CRAF by pan-RAF inhibitors plays a key role in determining cellular response. Importantly, in contrast to the modest activity seen with single-agent treatment, dual pan-RAF and MEK inhibition results in the induction of apoptosis, greatly enhancing efficacy. Notably, combined pan-RAF and MEK inhibition can overcome intrinsic and acquired resistance to single-agent RAF/MEK inhibition, supporting dual pan-RAF and MEK inhibition as a novel therapeutic strategy for BRAF- and KRAS-mutant cancers.
KRAS-mutation is a key driver of pancreatic cancer and PI3K pathway activity is an additional requirement for Kras-induced tumorigenesis. Clinical trials of PI3K pathway inhibitors in pancreatic ...cancer have shown limited responses. Understanding the molecular basis for this lack of efficacy may direct future treatment strategies with emerging PI3K inhibitors. We sought new therapeutic approaches that synergise with PI3K inhibitors through pooled CRISPR modifier genetic screening and a drug combination screen. ERBB-family receptor tyrosine kinase signalling and mTOR signalling were key modifiers of sensitivity to alpelisib and pictilisib. Inhibition of the ERBB-family or mTOR was synergistic with PI3K inhibition in spheroid, stromal co-cultures. Near-complete loss of ribosomal S6 phosphorylation was associated with synergy. Genetic alterations in the ERBB-PI3K signalling axis were associated with decreased survival of pancreatic cancer patients. Suppression of the PI3K/mTOR axis is potentiated by dual PI3K and ERBB family or mTOR inhibition. Surprisingly, despite the presence of oncogenic KRAS, thought to bestow independence from receptor tyrosine kinase signalling, inhibition of the ERBB family blocks downstream pathway activation and synergizes with PI3K inhibitors. Further exploration of these therapeutic combinations is warranted for the treatment of pancreatic cancer.
Abstract
Through the use of an unbiased, genome-scale CRISPR modifier screen, we identified NF1 suppression as a mechanism of resistance to EGFR inhibition in NRAS/KRAS/BRAFV600-wild-type colorectal ...cancer cells. Reduced NF1 expression permitted sustained signaling through the MAPK pathway to promote cell proliferation in the presence of EGFR inhibition. Targeting of MEK in combination with EGFR inhibition leads to synergistic antiproliferative activity. Human KRAS/NRAS/BRAFV600-wild-type colorectal cancer cell lines with NF1 mutations displayed reduced NF1 mRNA or protein expression and were resistant to EGFR blockade by gefitinib or cetuximab. Cooccurring loss-of-function mutations in PTEN were associated with resistance to dual EGFR/MEK inhibition but cotreatment with a PI3K inhibitor further suppressed proliferation. Loss of NF1 may be a useful biomarker to identify patients that are less likely to benefit from single-agent anti-EGFR therapy in colorectal cancer and may direct potential combination strategies.
Implications:
This study suggests that further clinical validation of NF1 status as predictor of response to anti-EGFR targeting antibodies in patients with colorectal cancer with KRAS/NRAS/BRAFV600-wild-type tumors is warranted.
Multiple myeloma (MM) remains incurable despite recent therapeutic advances. RAS mutations are frequently associated with relapsed/refractory disease. Efforts to target the mitogen-activated protein ...kinase (MAPK) pathway with the MEK inhibitor, trametinib (Tra) have been limited by toxicities and the development of resistance. Dexamethasone (Dex) is a corticosteroid commonly used in clinical practice, to enhance efficacy of anti-myeloma therapy. Therefore, we hypothesised that the combination of Tra and Dex would yield synergistic activity in RAS-mutant MM.
The response of human MM cell lines to drug treatment was analysed using cell proliferation assays, Western blotting, Annexin V and propidium iodide staining by flow cytometry and reverse phase protein arrays. The efficacy of trametinib and dexamethasone treatment in the MM.1S xenograft model was assessed by measuring tumor volume over time.
The Tra/Dex combination demonstrated synergistic cytotoxicity in KRAS
mutant lines MM.1S and RPMI-8226. The induction of apoptosis was associated with decreased MCL-1 expression and increased BIM expression. Reverse phase proteomic arrays revealed suppression of FAK, PYK2, FLT3, NDRG1 and 4EBP1 phosphorylation with the Tra/Dex combination. Notably, NDRG1 expression was associated with the synergistic response to Tra/Dex. MM cells were sensitive to PDK1 inhibition and IGF1-induced signalling partially protected from Tra/Dex treatment, highlighting the importance of this pathway. In the MM.1S tumor xenograft model, only the combination of Tra/Dex resulted in a significant inhibition of tumor growth.
Overall Tra/Dex demonstrates antiproliferative activity in RAS-mutant MM cell lines associated with suppression of pro-survival PDK1 signalling and engagement of apoptotic pathways. Our data support further investigation of this combination in RAS-mutant MM.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
BRAF inhibitors, such as vemurafenib, have shown efficacy in BRAF-mutant melanoma treatment but acquired-resistance invariably develops. Unveiling the potential vulnerabilities associated with ...vemurafenib resistance could provide rational strategies for combinatorial treatment.
This work investigates the metabolic characteristics and vulnerabilities of acquired resistance to vemurafenib in three generated BRAF-mutant human melanoma cell clones, analysing metabolic profiles, gene and protein expression in baseline and nutrient withdrawal conditions. Preclinical findings are correlated with gene expression analysis from publicly available clinical datasets.
Two vemurafenib-resistant clones showed dependency on lipid metabolism and increased prostaglandin E2 synthesis and were more responsive to vemurafenib under EGFR inhibition, potentially implicating inflammatory lipid and EGFR signalling in ERK reactivation and vemurafenib resistance. The third resistant clone showed higher pyruvate-carboxylase (PC) activity indicating increased anaplerotic mitochondrial metabolism, concomitant with reduced GLUT-1, increased PC protein expression and survival advantage under nutrient-depleted conditions. Prostaglandin synthase (PTGES) expression was inversely correlated with melanoma patient survival. Increases in PC and PTGES gene expression were observed in some patients following progression on BRAF inhibitors.
Altogether, our data highlight heterogeneity in metabolic adaptations during acquired resistance to vemurafenib in BRAF-mutant melanoma, potentially uncovering key clinically-relevant mechanisms for combinatorial therapeutic targeting.
Deregulation of the cyclin‐dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the ...treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X‐ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small‐molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer.
CCT068127 is a novel inhibitor of CDK2 and CDK9, which achieves potent inhibition of RB and RNA polymerase II phosphorylation in human colon cancer cells. MCL1 protein levels were rapidly decreased by CCT068127 treatment and associated with synergistic activity with the combination of CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer.
Deregulation of the cell cycle commonly occurs during tumorigenesis, resulting in unrestricted cell proliferation and independence from mitogens. Cyclin-dependent kinase inhibitors have the potential ...to induce cell cycle arrest and apoptosis in cancer cells. CYC202 (R-roscovitine) is a potent inhibitor of CDK2/cyclin E that is undergoing clinical trials. Drugs selected to act on a particular molecular target may exert additional or alternative effects in intact cells. We therefore studied the molecular pharmacology of CYC202 in human colon cancer cells. Treatment of HT29 and KM12 colon carcinoma cell lines with CYC202 decreased both retinoblastoma protein phosphorylation and total retinoblastoma protein. In addition, an increase in the phosphorylation of extracellular signal-regulated kinases 1/2 was observed. As a result, downstream activation of the mitogen-activated protein kinase pathway occurred, as demonstrated by an increase in ELK-1 phosphorylation and in c-FOS expression. Use of mitogen-activated protein kinase kinases 1/2 inhibitors showed that the CYC202-induced extracellular signal-regulated kinases 1/2 phosphorylation was mitogen-activated protein kinase kinases 1/2 dependent but did not contribute to the cell cycle effects of the drug, which included a reduction of cells in G(1), inhibition of bromodeoxyuridine incorporation during S-phase, and a moderate increase in G(2)-M phase. Despite activation of the mitogen-activated protein kinase pathway, cyclin D1 protein levels were decreased by CYC202, an effect that occurred simultaneously with loss of retinoblastoma protein phosphorylation and inhibition of cell cycle progression. The reduced expression of cyclin D1 protein was independent of the p38(SAPK) and phosphatidylinositol 3-kinase pathways, which are known regulators of cyclin D1 protein. Interestingly, CYC202 caused a clear reduction in cyclins D1, A, and B1 mRNA, whereas c-FOS mRNA increased by 2-fold. This was accompanied by a loss of RNA polymerase II phosphorylation and total RNA polymerase II protein, suggesting that CYC202 was inhibiting transcription, possibly via inhibition of CDK7 and CDK9 complexes. It can be concluded that although CYC202 can act as a CDK2 inhibitor, it also has the potential to inhibit CDK4 and CDK1 activities in cancer cells through the down-regulation of the corresponding cyclin partners. This provides a possible mechanism by which CYC202 can cause a reduction in retinoblastoma protein phosphorylation at multiple sites and cell cycle arrest in G(1), S, and G(2)-M phases. In addition to providing useful insights into the molecular pharmacology of CYC202 in human cancer cells, the results also suggest potential pharmacodynamic end points for use in clinical trials with the drug.
Despite showing clinical activity in BRAF-mutant melanoma, the MEK inhibitor (MEKi) trametinib has failed to show clinical benefit in KRAS-mutant colorectal cancer. To identify mechanisms of ...resistance to MEKi, we employed a pharmacogenomic analysis of MEKi-sensitive versus MEKi-resistant colorectal cancer cell lines. Strikingly, interferon- and inflammatory-related gene sets were enriched in cell lines exhibiting intrinsic and acquired resistance to MEK inhibition. The bromodomain inhibitor JQ1 suppressed interferon-stimulated gene (ISG) expression and in combination with MEK inhibitors displayed synergistic effects and induced apoptosis in MEKi-resistant colorectal cancer cell lines. ISG expression was confirmed in patient-derived organoid models, which displayed resistance to trametinib and were resensitized by JQ1 co-treatment. In in vivo models of colorectal cancer, combination treatment significantly suppressed tumor growth. Our findings provide a novel explanation for the limited response to MEK inhibitors in KRAS-mutant colorectal cancer, known for its inflammatory nature. Moreover, the high expression of ISGs was associated with significantly reduced survival of colorectal cancer patients. Excitingly, we have identified novel therapeutic opportunities to overcome intrinsic and acquired resistance to MEK inhibition in colorectal cancer.
PDF
