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Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E. When phosphorylated, eIF4E specifically engenders the translation of mRNAs that code for proteins involved in cell survival. We hypothesized that cancer cells treated with palbociclib use upregulated phosphorylated eIF4E (phospho-eIF4E) to escape the antitumor benefits of this drug. Indeed, we found that pharmacologic or genetic disruption of MNK1/2 activity, the only known kinases for eIF4E, enhanced the ability of palbociclib to decrease clonogenic outgrowth. Moreover, a quantitative proteomics analysis of melanoma cells treated with combined MNK1/2 and CDK4/6 inhibitors showed downregulation of proteins with critical roles in cell-cycle progression and mitosis, including AURKB, TPX2, and survivin. We also observed that palbociclib-resistant breast cancer cells have higher basal levels of phospho-eIF4E, and that treatment with MNK1/2 inhibitors sensitized these palbociclib-resistant cells to CDK4/6 inhibition. In vivo we demonstrate that the combination of MNK1/2 and CDK4/6 inhibition significantly increases the overall survival of mice compared with either monotherapy. Overall, our data support MNK1/2 inhibitors as promising drugs to potentiate the antineoplastic effects of palbociclib and overcome therapy-resistant disease.

Supplementary Figures S1-S5

Supplementary Table S1

Combined inhibition of BRAF, MEK, and CDK4/6 is currently under evaluation in clinical trials for patients with melanoma harboring a BRAFV600 mutation. While this triple therapy has potent tumor-intrinsic effects, the impact of this combination on antitumor immunity remains unexplored. Here, using a syngeneic BrafV600ECdkn2a−/−Pten−/− melanoma model, we demonstrated that triple therapy promoted durable tumor control through tumor-intrinsic mechanisms and promoted immunogenic cell death and T-cell infiltration. Despite this, tumors treated with triple therapy were unresponsive to immune checkpoint blockade (ICB). Flow cytometric and single-cell RNA sequencing analyses of tumor-infiltrating immune populations revealed that triple therapy markedly depleted proinflammatory macrophages and cross-priming CD103+ dendritic cells, the absence of which correlated with poor overall survival and clinical responses to ICB in patients with melanoma. Indeed, immune populations isolated from tumors of mice treated with triple therapy failed to stimulate T-cell responses ex vivo. While combined BRAF, MEK, and CDK4/6 inhibition demonstrates favorable tumor-intrinsic activity, these data suggest that collateral effects on tumor-infiltrating myeloid populations may impact antitumor immunity. These findings have important implications for the design of combination strategies and clinical trials that incorporate BRAF, MEK, and CDK4/6 inhibition with immunotherapy for the treatment of patients with melanoma.

All Supplementary Methods and associated references

PDF file 658K, Supplementary Methods

Supplementary Table S1

MetaCore analysis results

Aberrant cell-cycle progression is characteristic of melanoma, and CDK4/6 inhibitors, such as palbociclib, are currently being tested for efficacy in this disease. Despite the promising nature of CDK4/6 inhibitors, their use as single agents in melanoma has shown limited clinical benefit. Herein, we discovered that treatment of tumor cells with palbociclib induces the phosphorylation of the mRNA translation initiation factor eIF4E. When phosphorylated, eIF4E specifically engenders the translation of mRNAs that code for proteins involved in cell survival. We hypothesized that cancer cells treated with palbociclib use upregulated phosphorylated eIF4E (phospho-eIF4E) to escape the antitumor benefits of this drug. Indeed, we found that pharmacologic or genetic disruption of MNK1/2 activity, the only known kinases for eIF4E, enhanced the ability of palbociclib to decrease clonogenic outgrowth. Moreover, a quantitative proteomics analysis of melanoma cells treated with combined MNK1/2 and CDK4/6 inhibitors showed downregulation of proteins with critical roles in cell-cycle progression and mitosis, including AURKB, TPX2, and survivin. We also observed that palbociclib-resistant breast cancer cells have higher basal levels of phospho-eIF4E, and that treatment with MNK1/2 inhibitors sensitized these palbociclib-resistant cells to CDK4/6 inhibition. In vivo we demonstrate that the combination of MNK1/2 and CDK4/6 inhibition significantly increases the overall survival of mice compared with either monotherapy. Overall, our data support MNK1/2 inhibitors as promising drugs to potentiate the antineoplastic effects of palbociclib and overcome therapy-resistant disease.

RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) is tightly regulated downstream of oncogenic pathways, and its dysregulation is a common feature in cancer. We evaluated CX-5461, the first-in-class selective rDNA transcription inhibitor, in a first-in-human, phase I dose-escalation study in advanced hematologic cancers. Administration of CX-5461 intravenously once every 3 weeks to 5 cohorts determined an MTD of 170 mg/m2, with a predictable pharmacokinetic profile. The dose-limiting toxicity was palmar–plantar erythrodysesthesia; photosensitivity was a dose-independent adverse event (AE), manageable by preventive measures. CX-5461 induced rapid on-target inhibition of rDNA transcription, with p53 activation detected in tumor cells from one patient achieving a clinical response. One patient with anaplastic large cell lymphoma attained a prolonged partial response and 5 patients with myeloma and diffuse large B-cell lymphoma achieved stable disease as best response. CX-5461 is safe at doses associated with clinical benefit and dermatologic AEs are manageable.Significance:CX-5461 is a first-in-class selective inhibitor of rDNA transcription. This first-in-human study establishes the feasibility of targeting this process, demonstrating single-agent antitumor activity against advanced hematologic cancers with predictable pharmacokinetics and a safety profile allowing prolonged dosing. Consistent with preclinical data, antitumor activity was observed in TP53 wild-type and mutant malignancies.This article is highlighted in the In This Issue feature, p. 983

Identification of genomic alterations defining ovarian carcinoma subtypes may aid the stratification of patients to receive targeted therapies. We characterized high-grade serous ovarian carcinoma (HGSC) for the association of amplified and overexpressed genes with clinical outcome using gene expression data from 499 HGSC patients in the Ovarian Tumor Tissue Analysis cohort for 11 copy number amplified genes: ATP13A4, BMP8B, CACNA1C, CCNE1, DYRK1B, GAB2, PAK4, RAD21, TPX2, ZFP36, and URI. The Australian Ovarian Cancer Study and The Cancer Genome Atlas datasets were also used to assess the correlation between gene expression, patient survival, and tumor classification. In a multivariate analysis, high GAB2 expression was associated with improved overall and progression-free survival (P = 0.03 and 0.02), whereas high BMP8B and ATP13A4 were associated with improved progression-free survival (P = 0.004 and P = 0.02). GAB2 overexpression and copy number gain were enriched in the AOCS C4 subgroup. High GAB2 expression correlated with enhanced sensitivity in vitro to the dual PI3K/mTOR inhibitor PF-04691502 and could be used as a genomic marker for identifying patients who will respond to treatments inhibiting PI3K signaling. Mol Cancer Ther; 14(6); 1495–503. ©2015 AACR.

XLS file 118K, Table listing gene sets identified by GSEA as significantly enriched in control versus vemurafenib treated A375 cells (FDR>0.25)

Exposure-response relationship between CX-5461 levels and rDNA transcription rate in normal PBMCs

Supplementary Figure 2

Supplementary Figure 3

All Supplementary Tables

Supplementary Figures 1-12

PDF file 3260K, Supplementary figures and tables

List of differentially expressed proteins annotated by cluster with pathway analysis in BLM cells

Supplementary Figure 1

Nanostring data including clinical information

Supplementary Figures S1 (Nanostring data and combined PFS for BMP8B and ATP13A4), S2 (Nanostring expression histograms), S3 (GAB2 survival by cohort), S4 (BMP8B and ATP13A4 survival by cohort), S5 (Metacore network maps); Supplementary Tables S2 (Cell line information), S3 (Univariate survival analysis results), S4 (Step-wise AIC results) and S6 (association of GAB2 expression with PRAS40).

XLS file 72K, Table describing the 300 genes whose mRNA expression were most significantly changed by vemurafenib treatment in A375 cells

PDF file - 68K, Z = -1.5 and corresponding loss of viability for all cell lines

PDF file - 68K, Cell line culture and transfection conditions

PDF file - 126K, TPX2 amplification is protective against loss of cellular viability in ovarian tumor cells

Purpose: Ovarian cancer has the highest mortality rate of all the gynecologic malignancies and is responsible for approximately 140,000 deaths annually worldwide. Copy number amplification is frequently associated with the activation of oncogenic drivers in this tumor type, but their cytogenetic complexity and heterogeneity has made it difficult to determine which gene(s) within an amplicon represent(s) the genuine oncogenic driver. We sought to identify amplicon targets by conducting a comprehensive functional analysis of genes located in the regions of amplification in high-grade serous and endometrioid ovarian tumors.Experimental Design: High-throughput siRNA screening technology was used to systematically assess all genes within regions commonly amplified in high-grade serous and endometrioid cancer. We describe the results from a boutique siRNA screen of 272 genes in a panel of 18 ovarian cell lines. Hits identified by the functional viability screen were further interrogated in primary tumor cohorts to determine the clinical outcomes associated with amplification and gene overexpression.Results: We identified a number of genes as critical for cellular viability when amplified, including URI1, PAK4, GAB2, and DYRK1B. Integration of primary tumor gene expression and outcome data provided further evidence for the therapeutic use of such genes, particularly URI1 and GAB2, which were significantly associated with survival in 2 independent tumor cohorts.Conclusion: By taking this integrative approach to target discovery, we have streamlined the translation of high-resolution genomic data into preclinical in vitro studies, resulting in the identification of a number of genes that may be specifically targeted for the treatment of advanced ovarian tumors. Clin Cancer Res; 19(6); 1411–21. ©2013 AACR.

PDF file - 155K, Screen control metrics

PDF file - 89K, Boutique siRNA library

PDF file - 75K, Oligonucleotides

PDF file - 81K, All screening hits

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PDF file - 119K, Supp Table S1. Regions of differential copy number change between primary tumor samples and most distant abdominal deposit Supp Table S2. Top 50 pathways altered between primary tumors and abdominal deposits Supp Table S3. Individual patient chemotherapy regimens for paired primary and relapse ovarian tumors Supp Table S4. BRCA mutation details for mutation positive cases Supp Table S5. Regions of discrete copy number amplification Supp Table S6. Regions of discrete copy number deletion

Pharmacologic inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) are an approved treatment for hormone receptor–positive breast cancer and are currently under evaluation across hundreds of clinical trials for other cancer types. The clinical success of these inhibitors is largely attributed to well-defined tumor-intrinsic cytostatic mechanisms, whereas their emerging role as immunomodulatory agents is less understood. Using integrated epigenomic, transcriptomic, and proteomic analyses, we demonstrated a novel action of CDK4/6 inhibitors in promoting the phenotypic and functional acquisition of immunologic T-cell memory. Short-term priming with a CDK4/6 inhibitor promoted long-term endogenous antitumor T-cell immunity in mice, enhanced the persistence and therapeutic efficacy of chimeric antigen receptor T cells, and induced a retinoblastoma-dependent T-cell phenotype supportive of favorable responses to immune checkpoint blockade in patients with melanoma. Together, these mechanistic insights significantly broaden the prospective utility of CDK4/6 inhibitors as clinical tools to boost antitumor T-cell immunity. Significance: Immunologic memory is critical for sustained antitumor immunity. Our discovery that CDK4/6 inhibitors drive T-cell memory fate commitment sheds new light on their clinical activity, which is essential for the design of clinical trial protocols incorporating these agents, particularly in combination with immunotherapy, for the treatment of cancer. This article is highlighted in the In This Issue feature, p. 2355

Targeted and immune therapies have transformed outcomes for melanoma patients, nevertheless, significant challenges remain to maximize their clinical benefit. BRAF/MEK targeted therapy (TT) is associated with very high initial response rates that are typically short-lived, while in contrast Immune Checkpoint Inhibitors (ICI) provide more durable responses but have lower initial response rates. Hence, it has been proposed that sequencing ICIs with TT would lead to a higher frequency of durable responses. However, there are many unanswered questions regarding the choice of immunotherapy, the best sequencing strategy and the mechanism leading to this synergy. To address these questions, we have optimized a syngeneic mouse melanoma model, YUMMER1.7-PV1 that is sensitive to both TT and ICIs. Importantly, this model recapitulates the known three therapeutic response phases of targeted therapies: initial response, followed by drug tolerance and then resistance. Investigation of the tumor immune microenvironment (TIME) at these three drug-induced phases, demonstrates that the immune suppressive microenvironment present during the acquired resistant phase is primed at the drug tolerance phase. Utilizing this model, we have explored the time-dependent therapy-induced changes in both tumor cells and the TIME during various TT and ICI sequencing strategies. This information will determine the optimal sequencing strategy of targeted therapy and immunotherapy that will deliver better outcomes for melanoma patients. Citation Format: Riyaben Patel, Anna Trigos, Nicole Haynes, Emily Lelliott, Grant McArthur, Karen E. Sheppard. Sequencing of targeted- and immune-therapy: delineating time dependent changes in both melanoma cells and the immune microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5941.

Resistance to targeted therapies is a major challenge in cancer care and occurs via genetic and non-genetic mechanisms. In this issue of Cancer Cell, Marin-Bejar et al. demonstrate that melanomas recurrently select genetic or non-genetic resistance trajectories and that targeting neural crest stem cell-like cells prevents non-genetic, but not genetic, resistance.

Background Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. Methods Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. Results We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. Conclusions Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease., Acquired resistance limits the efficacy of PARP inhibitors (PARPi) in high grade serous ovarian cancer (HGSOC). Here, the authors show that inhibition of RNA polymerase I transcription using CX-5461 increases the therapeutic efficacy of PARPi and overcomes PARPi resistance in PDX models of HGSOC.

Simple Summary Targeted anti-cancer therapies have revolutionised melanoma patient care; however, cures remain uncommon due to acquired drug resistance that results in disease relapse. Recent insights from the clinic and experimental settings have identified a key role for metabolic plasticity, defined as the flexibility to utilise different nutrients and process them in different ways, in both disease progression and response to targeted therapies. Here, we discuss how this plasticity creates a moving target with important implications for identifying new combination therapies. Abstract Resistance to therapy continues to be a barrier to curative treatments in melanoma. Recent insights from the clinic and experimental settings have highlighted a range of non-genetic adaptive mechanisms that contribute to therapy resistance and disease relapse, including transcriptional, post-transcriptional and metabolic reprogramming. A growing body of evidence highlights the inherent plasticity of melanoma metabolism, evidenced by reversible metabolome alterations and flexibility in fuel usage that occur during metastasis and response to anti-cancer therapies. Here, we discuss how the inherent metabolic plasticity of melanoma cells facilitates both disease progression and acquisition of anti-cancer therapy resistance. In particular, we discuss in detail the different metabolic changes that occur during the three major phases of the targeted therapy response—the early response, drug tolerance and acquired resistance. We also discuss how non-genetic programs, including transcription and translation, control this process. The prevalence and diverse array of these non-genetic resistance mechanisms poses a new challenge to the field that requires innovative strategies to monitor and counteract these adaptive processes in the quest to prevent therapy resistance.

Simple Summary The burden of pancreatic ductal adenocarcinoma (PDAC) increases with rising incidence, yet 5-year overall survival remains poor at 17%. Routine comprehensive genomic profiling of PDAC only finds 2.5% of patients who may benefit and receive matched targeted therapy. Protein arginine methyltransferase 5 (PRMT5) as an anti-cancer target has gained significant interest in recent years and high levels of PRMT5 protein are associated with worse survival outcomes across multiple cancer types. Inhibition of PRMT5 in pre-clinical models can lead to cancer growth inhibition. However, PRMT5 is involved in multiple cellular processes, thus determining its mechanism of action is challenging. While past reviews on PRMT5 have focused on its role in diverse cellular processes and past research studies have focused mainly on haematological malignancies and glioblastoma, this review provides an overview of the possible biological mechanisms of action of PRMT5 inhibition and its potential as a treatment in pancreatic cancer. Abstract The overall survival of pancreatic ductal adenocarcinoma (PDAC) remains poor and its incidence is rising. Targetable mutations in PDAC are rare, thus novel therapeutic approaches are needed. Protein arginine methyltransferase 5 (PRMT5) overexpression is associated with worse survival and inhibition of PRMT5 results in decreased cancer growth across multiple cancers, including PDAC. Emerging evidence also suggests that altered RNA processing is a driver in PDAC tumorigenesis and creates a partial dependency on this process. PRMT5 inhibition induces altered splicing and this vulnerability can be exploited as a novel therapeutic approach. Three possible biological pathways underpinning the action of PRMT5 inhibitors are discussed; c-Myc regulation appears central to its action in the PDAC setting. Whilst homozygous MTAP deletion and symmetrical dimethylation levels are associated with increased sensitivity to PRMT5 inhibition, neither measure robustly predicts its growth inhibitory response. The immunomodulatory effect of PRMT5 inhibitors on the tumour microenvironment will also be discussed, based on emerging evidence that PDAC stroma has a significant bearing on disease behaviour and response to therapy. Lastly, with the above caveats in mind, current knowledge gaps and the implications and rationales for PRMT5 inhibitor development in PDAC will be explored.

BackgroundMelanoma brain metastases (MBMs) are a challenging clinical problem with high morbidity and mortality. Although first-line dabrafenib–trametinib and ipilimumab–nivolumab have similar intracranial response rates (50%–55%), central nervous system (CNS) resistance to BRAF-MEK inhibitors (BRAF-MEKi) usually occurs around 6 months, and durable responses are only seen with combination immunotherapy. We sought to investigate the utility of ipilimumab–nivolumab after MBM progression on BRAF-MEKi and identify mechanisms of resistance.MethodsPatients who received first-line ipilimumab–nivolumab for MBMs or second/third line ipilimumab–nivolumab for intracranial metastases with BRAFV600 mutations with prior progression on BRAF-MEKi and MRI brain staging from March 1, 2015 to June 30, 2018 were included. Modified intracranial RECIST was used to assess response. Formalin-fixed paraffin-embedded samples of BRAFV600 mutant MBMs that were naïve to systemic treatment (n=18) or excised after progression on BRAF-MEKi (n=14) underwent whole transcriptome sequencing. Comparative analyses of MBMs naïve to systemic treatment versus BRAF-MEKi progression were performed.ResultsTwenty-five and 30 patients who received first and second/third line ipilimumab–nivolumab, were included respectively. Median sum of MBM diameters was 13 and 20.5 mm for the first and second/third line ipilimumab–nivolumab groups, respectively. Intracranial response rate was 75.0% (12/16), and median progression-free survival (PFS) was 41.6 months for first-line ipilimumab–nivolumab. Efficacy of second/third line ipilimumab-nivolumab after BRAF-MEKi progression was poor with an intracranial response rate of 4.8% (1/21) and median PFS of 1.3 months. Given the poor activity of ipilimumab–nivolumab after BRAF-MEKi MBM progression, we performed whole transcriptome sequencing to identify mechanisms of drug resistance. We identified a set of 178 differentially expressed genes (DEGs) between naïve and MBMs with progression on BRAF-MEKi treatment (p value ConclusionsSecond-line ipilimumab–nivolumab for MBMs after BRAF-MEKi progression has poor activity. MBMs that are resistant to BRAF-MEKi that also conferred resistance to second-line ipilimumab–nivolumab showed enrichment of the IPRES gene signature.

Significance Targeting CDK4/6 shows great promise in the treatment of many solid cancers, including melanoma. This study has uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. In melanoma, palbociclib activates p53 via modulating PRMT5-dependent alternate MDM4 pre-mRNA splicing, which results in a robust decrease in MDM4 protein expression. Loss of palbociclib regulation of the PRMT5–MDM4 axis leads to drug resistance. Dual inhibition of CDK4/6 and PRMT5 potently suppresses melanoma tumor growth and is well tolerated in vivo. Our findings not only have immediate implications for the advancement of CDK4/6 inhibition for treating melanoma, but also more generally offer insights into CDK4/6 mechanism of action., Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5–MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5–MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.