Ribosome biogenesis and protein synthesis are fundamental rate-limiting steps for cell growth and proliferation. The ribosomal proteins (RPs), comprising the structural parts of the ribosome, are ...essential for ribosome assembly and function. In addition to their canonical ribosomal functions, multiple RPs have extra-ribosomal functions including activation of p53-dependent or p53-independent pathways in response to stress, resulting in cell cycle arrest and apoptosis. Defects in ribosome biogenesis, translation, and the functions of individual RPs, including mutations in RPs have been linked to a diverse range of human congenital disorders termed ribosomopathies. Ribosomopathies are characterized by tissue-specific phenotypic abnormalities and higher cancer risk later in life. Recent discoveries of somatic mutations in RPs in multiple tumor types reinforce the connections between ribosomal defects and cancer. In this article, we review the most recent advances in understanding the molecular consequences of RP mutations and ribosomal defects in ribosomopathies and cancer. We particularly discuss the molecular basis of the transition from hypo- to hyper-proliferation in ribosomopathies with elevated cancer risk, a paradox termed "Dameshek's riddle." Furthermore, we review the current treatments for ribosomopathies and prospective therapies targeting ribosomal defects. We also highlight recent advances in ribosome stress-based cancer therapeutics. Importantly, insights into the mechanisms of resistance to therapies targeting ribosome biogenesis bring new perspectives into the molecular basis of cancer susceptibility in ribosomopathies and new clinical implications for cancer therapy.
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.
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.
The nucleoli are subdomains of the nucleus that form around actively transcribed ribosomal RNA (rRNA) genes. They serve as the site of rRNA synthesis and processing, and ribosome assembly. There are ...400-600 copies of rRNA genes (rDNA) in human cells and their highly repetitive and transcribed nature poses a challenge for DNA repair and replication machineries. It is only in the last 7 years that the DNA damage response and processes of DNA repair at the rDNA repeats have been recognized to be unique and distinct from the classic response to DNA damage in the nucleoplasm. In the last decade, the nucleolus has also emerged as a central hub for coordinating responses to stress via sequestering tumor suppressors, DNA repair and cell cycle factors until they are required for their functional role in the nucleoplasm. In this review, we focus on features of the rDNA repeats that make them highly vulnerable to DNA damage and the mechanisms by which rDNA damage is repaired. We highlight the molecular consequences of rDNA damage including activation of the nucleolar DNA damage response, which is emerging as a unique response that can be exploited in anti-cancer therapy. In this review, we focus on CX-5461, a novel inhibitor of Pol I transcription that induces the nucleolar DNA damage response and is showing increasing promise in clinical investigations.
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.
Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC.
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.
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.
The three-dimensional organization of the genome contributes to its maintenance and regulation. While chromosomal regions associate with nucleolar ribosomal RNA genes (rDNA), the biological ...significance of rDNA-genome interactions and whether they are dynamically regulated during disease remain unclear. rDNA chromatin exists in multiple inactive and active states and their transition is regulated by the RNA polymerase I transcription factor UBTF. Here, using a MYC-driven lymphoma model, we demonstrate that during malignant progression the rDNA chromatin converts to the open state, which is required for tumor cell survival. Moreover, this rDNA transition co-occurs with a reorganization of rDNA-genome contacts which correlate with gene expression changes at associated loci, impacting gene ontologies including B-cell differentiation, cell growth and metabolism. We propose that UBTF-mediated conversion to open rDNA chromatin during malignant transformation contributes to the regulation of specific gene pathways that regulate growth and differentiation through reformed long-range physical interactions with the rDNA.
Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We ...hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naïve, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug‐tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.
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Cutaneous melanoma is marked by high incidence of BRAF mutations and cyclin‐dependent kinase 4 (CDK4) deregulation. Thus, the combination of CDK4 inhibition with BRAF and MEK inhibition is a promising strategy toward improving melanoma survival. Here, addition of the CDK4/6 inhibitor palbociclib upfront with BRAF and MEK inhibitors blocked the development of drug resistance and produced robust, durable responses in BRAF mutant melanoma cells. The synergistic effects were lost when palbociclib was started after melanoma cells became resistant to BRAF inhibition. The findings provide a rationale for upfront first‐line treatment with CDK4/6, BRAF, and MEK inhibitors in melanoma patients.
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RNA polymerase I (Pol I) transcription of the ribosomal RNA (rRNA) genes, a rate-limiting step for growth and proliferation, is a highly regulated process downstream of many oncogenic pathways. ...Dysregulation of ribosome biogenesis is a feature of numerous cancers (Bywater Nat Rev 2013 ).
CX-5461, a selective inhibitor of Pol I transcription (Senhwa Biosciences, San Diego, USA), has ~200-fold selectivity for inhibition of Pol I over Pol II (Drygin Can Res 2011 ). Inhibition of Pol I transcription by CX-5461 induces a p53 independent nucleolar stress response and a nucleolar specific DNA damage response (Quin Oncotarget 2016 ). CX-5461 provides survival benefit in mouse models of lymphoma, myeloid leukemia and myeloma (Hein Blood 2017) .
Methods: We initiated a first in class, first in human, phase I dose escalation study of CX-5461 in adult patients with advanced hematologic cancers, with no standard therapeutic options, adequate organ function and performance status to determine maximum tolerated dose (MTD), safety, pharmacokinetic (PK) profile and antitumor activity.
CX-5461 was administered by 1 hour IV infusion 3 weekly. Dose escalations were planned in 7 cohorts (25 - 450 mg/m2), in an accelerated design, with change to a 3+3 design based on predefined toxicity criteria.
Inhibition of Pol I transcription rate was measured via RNA-FISH, quantitating the abundance of 47S pre-rRNA levels in peripheral blood mononuclear cells (PBMC) and tumor tissue, at various time points following cycle 1. Skin biopsies from normal skin and rash areas were studied after the observation of photosensitivity in patients in cohort 1.
Results: 16 patients (6 myeloma, 2 Hodgkin lymphoma, 6 Non Hodgkin lymphoma (NHL), 1 TPLL, 1 CLL) were treated in 5 cohorts (25 - 250 mg/ m2), for a median of 2 (1 - 18) cycles. The MTD was 170 mg/ m2. The dose limiting toxicity was palmar plantar erythrodysaesthesia in 2 patients at 250 mg/ m2. One of these patients continued treatment for 17 further cycles at 170 mg/ m2. Eight patients (50%) had grade </=3 treatment related photosensitivity across all dose cohorts, this did not recur on re-exposure with appropriate protection. No other grade 3+ treatment related adverse events were observed.
The average terminal half-life (T1/2) showed an increasing trend with dose escalation, in a profile consistent with enterohepatic recycling. The maximum T1/2 noted was 92 hours in cohort 5. Linear behaviors were generally observed in Cmax and AUC exposure parameters.
The best response seen was an excellent prolonged partial response in 1 patient with anaplastic large cell lymphoma (18 cycles) and stable disease in 3 patients with myeloma (4 - 6 cycles) and 2 with diffuse large B-cell lymphoma (4 - 16 cycles). Clinical and radiologic response was noted in an area of high grade transformation in a patient with cutaneous T-Cell lymphoma (CTCL).
Consistent, significant decreases in Pol I transcription were observed at 1hr post-infusion in PBMC. The average level of inhibition was 49.0% (22.9 - 69.9%), 51.1% (34.4 - 64.4%), 19.6% (-72.0 - 69.7%), 47.3% (46.5 - 48.0%) and 38.6% (6.8 - 70.4%) in cohorts 1 - 5 respectively (Fig 1). On target activity at 24hrs was observed in most tumor biopsies, where the level of inhibition was variable (10/13; median range: 4.8 - 68.9 %) (Fig 2).
Targeted exon sequencing determined TP53 mutational status in selected patients (n=13). 4 patients with mutations experienced early disease progression. In TP53 wildtype patients, 4 achieved periods of stable disease. In a CTCL patient, stabilization of p53 protein levels and increase in the p53 target gene p21 occurred in an area of high grade transformation where response was observed (Fig 3).
A spongiotic or parakeratosis reaction pattern was observed in biopsies from the photosensitivity rash, with increase in p53 IHC expression in the epidermis of normal skin following CX-5461 exposure (Fig 4).
Conclusion: Our first in human study of the Pol I inhibitor CX-5461, has determined a MTD of 170 mg/ m2 every 3 weeks, with a predictable PK profile. The drug is well tolerated and compatible with a prolonged duration of treatment. Photosensitivity can be a significant adverse event independent of dose, which is manageable by careful attention to preventive measures. Durable periods of response or stability have been noted in heavily pretreated chemorefractory patients with NHL and myeloma. Further studies to explore weekly dosing regimens are planned.
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Khot:Celgene: Consultancy; Janssen: Consultancy; Amgen: Other: Travel Grant. Lim:Senhwa Biosciences: Consultancy, Equity Ownership. Soong:Senwa Biosciences: Employment, Equity Ownership. Harrison:Celgene: Consultancy, Research Funding, Speakers Bureau.
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