Targeting CCNE1 amplified ovarian and endometrial cancers by combined inhibition of PKMYT1 and ATR.

Ovarian cancers (OVCAs) and endometrial cancers (EMCAs) with CCNE1-amplification are often resistant to standard of care treatment and represent an unmet clinical need. Previously, synthetic-lethal screening identified loss of the CDK1 regulator, PKMYT1, as synthetically lethal with CCNE1-amplification. We hypothesized that CCNE1-amplification associated replication stress will be more effectively targeted by combining the PKMYT1 inhibitor, lunresertib (RP-6306), with the ATR inhibitor, camonsertib (RP-3500/RG6526). Low dose combination RP-6306 with RP-3500 synergistically increased cytotoxicity more in CCNE1 amplified compared to non-amplified cells. Combination treatment produced durable antitumor activity and increased survival in CCNE1 amplified patient-derived and cell line-derived xenografts. Mechanistically, low doses of RP-6306 with RP-3500 increase CDK1 activation more so than monotherapy, triggering rapid and robust induction of premature mitosis, DNA damage and apoptosis in a CCNE1-dependent manner. These findings suggest that targeting CDK1 activity by combining RP-6306 with RP-3500 is a novel therapeutic approach to treat CCNE1-amplifed OVCAs and EMCAs.

Final Overall Survival Efficacy Results of Ivosidenib for Patients With Advanced Cholangiocarcinoma With IDH1 Mutation: The Phase 3 Randomized Clinical ClarIDHy Trial.

IMPORTANCE: Isocitrate dehydrogenase 1 (IDH1) variations occur in up to approximately 20% of patients with intrahepatic cholangiocarcinoma. In the ClarIDHy trial, progression-free survival as determined by central review was significantly improved with ivosidenib vs placebo. OBJECTIVE: To report the final overall survival (OS) results from the ClarIDHy trial, which aimed to demonstrate the efficacy of ivosidenib (AG-120)-a first-in-class, oral, small-molecule inhibitor of mutant IDH1-vs placebo for patients with unresectable or metastatic cholangiocarcinoma with IDH1 mutation. DESIGN, SETTING, AND PARTICIPANTS: This multicenter, randomized, double-blind, placebo-controlled, clinical phase 3 trial was conducted from February 20, 2017, to May 31, 2020, at 49 hospitals across 6 countries among patients aged 18 years or older with cholangiocarcinoma with IDH1 mutation whose disease progressed with prior therapy. INTERVENTIONS: Patients were randomized 2:1 to receive ivosidenib, 500 mg, once daily or matched placebo. Crossover from placebo to ivosidenib was permitted if patients had disease progression as determined by radiographic findings. MAIN OUTCOMES AND MEASURES: The primary end point was progression-free survival as determined by blinded independent radiology center (reported previously). Overall survival was a key secondary end point. The primary analysis of OS followed the intent-to-treat principle. Other secondary end points included objective response rate, safety and tolerability, and quality of life. RESULTS: Overall, 187 patients (median age, 62 years [range, 33-83 years]) were randomly assigned to receive ivosidenib (n = 126; 82 women [65%]; median age, 61 years [range, 33-80 years]) or placebo (n = 61; 37 women [61%]; median age, 63 years [range, 40-83 years]); 43 patients crossed over from placebo to ivosidenib. The primary end point of progression-free survival was reported elsewhere. Median OS was 10.3 months (95% CI, 7.8-12.4 months) with ivosidenib vs 7.5 months (95% CI, 4.8-11.1 months) with placebo (hazard ratio, 0.79 [95% CI, 0.56-1.12]; 1-sided P = .09). When adjusted for crossover, median OS with placebo was 5.1 months (95% CI, 3.8-7.6 months; hazard ratio, 0.49 [95% CI, 0.34-0.70]; 1-sided P < .001). The most common grade 3 or higher treatment-emergent adverse event (≥5%) reported in both groups was ascites (11 patients [9%] receiving ivosidenib and 4 patients [7%] receiving placebo). Serious treatment-emergent adverse events considered ivosidenib related were reported in 3 patients (2%). There were no treatment-related deaths. Patients receiving ivosidenib reported no apparent decline in quality of life compared with placebo. CONCLUSIONS AND RELEVANCE: This randomized clinical trial found that ivosidenib was well tolerated and resulted in a favorable OS benefit vs placebo, despite a high rate of crossover. These data, coupled with supportive quality of life data and a tolerable safety profile, demonstrate the clinical benefit of ivosidenib for patients with advanced cholangiocarcinoma with IDH1 mutation. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02989857.

Molecular and morphological changes induced by ivosidenib correlate with efficacy in mutant-IDH1 cholangiocarcinoma.

Background: IDH1 mutations occur in approximately 13% of intrahepatic cholangiocarcinomas (IHCCs). The oral, targeted, mutant IDH1 (mIDH1) inhibitor ivosidenib (AG-120) suppresses production of the oncometabolite D-2-hydroxyglutarate, promoting disease stabilization and improved progression-free survival (PFS) in mIDH1 IHCC. Materials & methods: Harnessing matched baseline and on-treatment biopsies, we investigate the potential mechanisms underlying ivosidenib's efficacy. Results: mIDH1 inhibition leads to decreased cytoplasm and expression of hepatocyte lineage markers in patients with prolonged PFS. These findings are accompanied by downregulation of biliary fate, cell cycle progression and AKT pathway activity. Conclusion: Ivosidenib stimulates a hepatocyte differentiation program in mIDH1 IHCC, a phenotype associated with clinical benefit. mIDH1 inhibition could be a paradigm for differentiation-based therapy in solid tumors. Clinical trial registration: NCT02073994 (ClinicalTrials.gov).

MAT2A Inhibition Blocks the Growth of MTAP-Deleted Cancer Cells by Reducing PRMT5-Dependent mRNA Splicing and Inducing DNA Damage.

The methylthioadenosine phosphorylase (MTAP) gene is located adjacent to the cyclin-dependent kinase inhibitor 2A (CDKN2A) tumor-suppressor gene and is co-deleted with CDKN2A in approximately 15% of all cancers. This co-deletion leads to aggressive tumors with poor prognosis that lack effective, molecularly targeted therapies. The metabolic enzyme methionine adenosyltransferase 2α (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We report the characterization of potent MAT2A inhibitors that substantially reduce levels of S-adenosylmethionine (SAM) and demonstrate antiproliferative activity in MTAP-deleted cancer cells and tumors. Using RNA sequencing and proteomics, we demonstrate that MAT2A inhibition is mechanistically linked to reduced protein arginine methyltransferase 5 (PRMT5) activity and splicing perturbations. We further show that DNA damage and mitotic defects ensue upon MAT2A inhibition in HCT116 MTAP-/- cells, providing a rationale for combining the MAT2A clinical candidate AG-270 with antimitotic taxanes.

Safety and activity of ivosidenib in patients with IDH1-mutant advanced cholangiocarcinoma: a phase 1 study.

BACKGROUND: Isocitrate dehydrogenase-1 (IDH1) is mutated in up to 25% of cholangiocarcinomas, especially intrahepatic cholangiocarcinoma. Ivosidenib is an oral, targeted inhibitor of mutant IDH1 (mIDH1) approved in the USA for the treatment of mIDH1 acute myeloid leukaemia in newly diagnosed patients ineligible for intensive chemotherapy and patients with relapsed or refractory disease. Ivosidenib is under clinical evaluation in a phase 1 study that aims to assess its safety and tolerability in patients with mIDH1 solid tumours. Here we report data for the mIDH1-cholangiocarcinoma cohort. METHODS: We did a phase 1 dose-escalation and expansion study of ivosidenib monotherapy in mIDH1 solid tumours at 12 clinical sites in the USA and one in France. The primary outcomes were safety, tolerability, maximum tolerated dose, and recommended phase 2 dose. Eligible patients had a documented mIDH1 tumour based on local testing, an Eastern Cooperative Oncology Group performance status of 0 or 1, one or more previous lines of therapy, and evaluable disease by Response Evaluation Criteria in Solid Tumors version 1.1. During dose escalation, ivosidenib was administered orally at 200-1200 mg daily in 28-day cycles in a standard 3 + 3 design; during expansion, patients received the selected dose on the basis of pharmacodynamic, pharmacokinetic, safety, and activity data from dose escalation. Safety and clinical activity analyses were reported for all patients with mIDH1-cholangiocarcinoma who were enrolled and received at least one dose of study treatment. Enrolment is complete, and the study is ongoing. This trial is registered at ClinicalTrials.gov, number NCT02073994. FINDINGS: Between March 14, 2014 and May 12, 2017, 73 patients with mIDH1-cholangiocarcinoma were enrolled and received ivosidenib. No dose-limiting toxicities were reported and maximum tolerated dose was not reached; 500 mg daily was selected for expansion. Common (≥20%) adverse events, regardless of cause, were fatigue (31 [42%]; two [3%] grade ≥3), nausea (25 [34%]; one [1%] grade ≥3), diarrhoea (23 [32%]), abdominal pain (20 [27%]; two [3%] grade ≥3), decreased appetite (20 [27%]; one [1%] grade ≥3), and vomiting (17 [23%]). Common grade 3 or worse adverse events were ascites (four [5%]) and anaemia (three [4%]); the only treatment-related grade 3 or worse adverse event in more than one patient was fatigue (two [3%]). Two (3%) patients had serious adverse events leading to on-treatment death (Clostridioides difficile infection and procedural haemorrhage); neither was assessed by the investigator as related to treatment. 46 (63%) patients had adverse events deemed related to ivosidenib, of which four (5%) were grade 3 or higher (two [3%] for fatigue; one [1%] each for decreased blood phosphorus and increased blood alkaline phosphatase). One serious adverse event was considered possibly related to treatment (grade 2 supraventricular extrasystoles). Four (5%; 95% CI 1·5-13·4) patients had a partial response. Median progression-free survival was 3·8 months (95% CI 3·6-7·3), 6-month progression-free survival was 40·1% (28·4-51·6), and 12-month progression-free survival was 21·8% (12·3-33·0). Median overall survival was 13·8 months (95% CI 11·1-29·3); however, data were censored for 48 patients (66%). INTERPRETATION: Ivosidenib might offer a well tolerated option for patients with mIDH1-cholangiocarcinoma. An ongoing, global phase 3 study is evaluating ivosidenib versus placebo in patients with previously treated nonresectable or metastatic mIDH1-cholangiocarcinoma. FUNDING: Agios Pharmaceuticals, Inc.

The combination of TPL2 knockdown and TNFα causes synthetic lethality via caspase-8 activation in human carcinoma cell lines.

Most normal and tumor cells are protected from tumor necrosis factor α (TNFα)-induced apoptosis. Here, we identify the MAP3 kinase tumor progression locus-2 (TPL2) as a player contributing to the protection of a subset of tumor cell lines. The combination of TPL2 knockdown and TNFα gives rise to a synthetic lethality phenotype via receptor-interacting serine/threonine-protein kinase 1 (RIPK1)-dependent and -independent mechanisms. Whereas wild-type TPL2 rescues the phenotype, its kinase-dead mutant does not. Comparison of the molecular events initiated by small interfering RNA for TPL2 (siTPL2) ± TNFα in treatment-sensitive and -resistant lines revealed that the activation of caspase-8, downstream of miR-21-5p and cFLIP, is the dominant TPL2-dependent event. More important, comparison of the gene expression profiles of all of the tested cell lines results in the clustering of sensitive and resistant lines into distinct groups, providing proof of principle for the feasibility of generating a predictive tool for treatment sensitivity.

HIF-1α induction during reperfusion avoids maladaptive repair after renal ischemia/reperfusion involving miR127-3p.

Ischemia/reperfusion (I/R) leads to Acute Kidney Injury. HIF-1α is a key factor during organ response to I/R. We previously demonstrated that HIF-1α is induced during renal reperfusion, after ischemia. Here we investigate the role of HIF-1α and the HIF-1α dependent mechanisms in renal repair after ischemia. By interference of HIF-1α in a rat model of renal I/R, we observed loss of expression and mis-localization of e-cadherin and induction of α-SMA, MMP-13, TGFß, and collagen I. Moreover, we demonstrate that HIF-1α inhibition promotes renal cell infiltrates by inducing IL-1ß, TNF-α, MCP-1 and VCAM-1, through NFkB activity. In addition, HIF-1α inhibition induced proximal tubule cells proliferation but it did not induce compensatory apoptosis, both in vivo. In vitro, HIF-1α knockdown in HK2 cells subjected to hypoxia/reoxygenation (H/R) promote cell entry into S phase, correlating with in vivo data. HIF-1α interference leads to downregulation of miR-127-3p and induction of its target gene Bcl6 in vivo. Moreover, modulation of miR-127-3p in HK2 cells subjected to H/R results in EMT regulation: miR127-3p inhibition promote loss of e-cadherin and induction of α-SMA and collagen I. In conclusion, HIF-1α induction during reperfusion is a protector mechanism implicated in a normal renal tissue repair after I/R.

A Pilot Study Identifying a Set of microRNAs As Precise Diagnostic Biomarkers of Acute Kidney Injury.

In the last decade, Acute Kidney Injury (AKI) diagnosis and therapy have not notably improved probably due to delay in the diagnosis, among other issues. Precocity and accuracy should be critical parameters in novel AKI biomarker discovery. microRNAs are key regulators of cell responses to many stimuli and they can be secreted to the extracellular environment. Therefore, they can be detected in body fluids and are emerging as novel disease biomarkers. We aimed to identify and validate serum miRNAs useful for AKI diagnosis and management. Using qRT-PCR arrays in serum samples, we determined miRNAs differentially expressed between AKI patients and healthy controls. Statistical and target prediction analysis allowed us to identify a panel of 10 serum miRNAs. This set was further validated, by qRT-PCR, in two independent cohorts of patients with relevant morbi-mortality related to AKI: Intensive Care Units (ICU) and Cardiac Surgery (CS). Statistical correlations with patient clinical parameter were performed. Our results demonstrated that the 10 selected miRNAs (miR-101-3p, miR-127-3p, miR-210-3p, miR-126-3p, miR-26b-5p, miR-29a-3p, miR-146a-5p, miR-27a-3p, miR-93-3p and miR-10a-5p) were diagnostic biomarkers of AKI in ICU patients, exhibiting areas under the curve close to 1 in ROC analysis. Outstandingly, serum miRNAs estimated before CS predicted AKI development later on, thus becoming biomarkers to predict AKI predisposition. Moreover, after surgery, the expression of the miRNAs was modulated days before serum creatinine increased, demonstrating early diagnostic value. In summary, we have identified a set of serum miRNAs as AKI biomarkers useful in clinical practice, since they demonstrate early detection and high diagnostic value and they recognize patients at risk.

microRNAs in the kidney: Novel biomarkers of Acute Kidney Injury

microRNAs are small, endogenous RNA molecules which are critical for a new step in the regulation of the gene expression. They have become the most critical biological mediators characterized in the last ten years. microRNAs participate in almost every cellular process, therefore their deregulation is associated with the development of a wide range of pathologies, including kidney diseases. Increasing evidence demonstrates that microRNAs are key regulators of the normal kidney function and development, but they are also at the basis of several renal diseases. Recent works have established that these molecules can be secreted to extracellular environments, enabling their detection in peripheral body fluids such as urine and serum. Moreover, circulating miRNAs detected in body fluids turn into suitable biomarkers of kidney diseases, including acute kidney injury. This new generation of renal biomarkers could have a great impact in the clinical practice, significantly contributing to improve patient management. In this review, we discuss over the implication of microRNAs in normal kidney function and homeostasis as well as the role of circulating miRNAs as novel biomarkers of kidney diseases, focusing on their potential usefulness in acute kidney injury management (AU)

Los microARN son pequeñas moléculas endógenas de ARN de vital importancia para la regulación de la expresión génica. Se han convertido en los mediadores biológicos más importantes que se han caracterizado en los últimos diez años. Participan en casi todos los procesos celulares, por lo que su desregulación está asociada al desarrollo de muchas patologías, entre las que se encuentran las renales. Existen cada vez más pruebas que demuestran que los microARN son reguladores claves de la función y el desarrollo renal, aunque también se encuentran en el origen de algunas enfermedades renales. Los estudios más recientes han concluido que estas moléculas pueden ser secretadas al exterior de la célula, lo que permite que puedan ser detectadas en fluidos periféricos como la orina y el suero. Además, los microARN circulantes detectados en los fluidos corporales pueden ser biomarcadores adecuados de las enfermedades renales, entre las que se incluye la lesión renal aguda. Esta nueva generación de biomarcadores renales podría tener consecuencias importantes para la práctica clínica, ya que podrían contribuir significativamente a la mejora del manejo de los pacientes. En este trabajo se revisa la implicación de los microARN en la homeostasis y la función renal y el papel de los microARN circulantes como nuevos biomarcadores de las enfermedades renales, centrándonos en su potencial utilidad para el manejo de la lesión renal aguda (AU)

MicroRNAs in the kidney: novel biomarkers of acute kidney injury.

microRNAs are small, endogenous RNA molecules which are critical for a new step in the regulation of the gene expression. They have become the most critical biological mediators characterized in the last ten years. microRNAs participate in almost every cellular process, therefore their deregulation is associated with the development of a wide range of pathologies, including kidney diseases. Increasing evidence demonstrates that microRNAs are key regulators of the normal kidney function and development, but they are also at the basis of several renal diseases. Recent works have established that these molecules can be secreted to extracellular environments, enabling their detection in peripheral body fluids such as urine and serum. Moreover, circulating miRNAs detected in body fluids turn into suitable biomarkers of kidney diseases, including acute kidney injury. This new generation of renal biomarkers could have a great impact in the clinical practice, significantly contributing to improve patient management. In this review, we discuss over the implication of microRNAs in normal kidney function and homeostasis as well as the role of circulating miRNAs as novel biomarkers of kidney diseases, focusing on their potential usefulness in acute kidney injury management.

miR-127 protects proximal tubule cells against ischemia/reperfusion: identification of kinesin family member 3B as miR-127 target.

Ischemia/reperfusion (I/R) is at the basis of renal transplantation and acute kidney injury. Molecular mechanisms underlying proximal tubule response to I/R will allow the identification of new therapeutic targets for both clinical settings. microRNAs have emerged as crucial and tight regulators of the cellular response to insults including hypoxia. Here, we have identified several miRNAs involved in the response of the proximal tubule cell to I/R. Microarrays and RT-PCR analysis of proximal tubule cells submitted to I/R mimicking conditions in vitro demonstrated that miR-127 is induced during ischemia and also during reperfusion. miR-127 is also modulated in a rat model of renal I/R. Interference approaches demonstrated that ischemic induction of miR-127 is mediated by Hypoxia Inducible Factor-1alpha (HIF-1α) stabilization. Moreover, miR-127 is involved in cell-matrix and cell-cell adhesion maintenance, since overexpression of miR-127 maintains focal adhesion complex assembly and the integrity of tight junctions. miR-127 also regulates intracellular trafficking since miR-127 interference promotes dextran-FITC uptake. In fact, we have identified the Kinesin Family Member 3B (KIF3B), involved in cell trafficking, as a target of miR-127 in rat proximal tubule cells. In summary, we have described a novel role of miR-127 in cell adhesion and its regulation by HIF-1α. We also identified for the first time KIF3B as a miR-127 target. Both, miR-127 and KIF3B appear as key mediators of proximal epithelial tubule cell response to I/R with potential al application in renal ischemic damage management.

Interaction between intestinal dendritic cells and bacteria translocated from the gut in rats with cirrhosis.

UNLABELLED: Cirrhosis with ascites is associated with a high rate of gut bacterial translocation (GBT) and spontaneous bacterial infections of enteric origin. We addressed the activation state and role of intestinal dendritic cells (DCs) in experimental ascitic cirrhosis and their relationship with GBT. Cirrhosis with ascites was CCl(4) induced in rats. To examine their activation state and functions, DCs (CD103(+) RT1B(+) CD3(-) CD45RA(-) ) were isolated from the intestinal lamina propria and mesenteric lymph nodes (MLNs), and the following parameters were determined by flow cytometry: surface antigen expression; spontaneous or lipopolysaccharide-stimulated tumor necrosis factor alpha (TNF-α) production; and in vitro capacity to phagocytose latex beads and to migrate toward the chemokine (C-C motif) ligand 21. GBT was defined as the growth of bacteria in MLNs culture. Bacterial DNA (Bact-DNA) in MLNs was identified by polymerase chain reaction. In rats with Bact-DNA in MLNs without GBT, intestinal and MLNs CD103(+) -DCs showed features of activation, expansion of the proinflammatory CD4(+) -DC subpopulation, augmented TNF-α production, and increased phagocytic and migratory capacities. In contrast, in rats with GBT, CD103(+) -DCs showed the absence of an activated phenotype, lowered TNF-α production, and relatively deficient phagocytosis and migration capacities. The CD103(+) -DC of rats without Bact-DNA in MLNs or GBT were similar to controls. In cirrhotic rats, bowel decontamination with antibiotics eliminated Bact-DNA in MLNs and GBT, normalized the activation state and functions of CD103(+) -DCs, and increased their TNF-α production. CONCLUSION: In experimental cirrhosis with ascites, continuous pressure of gut bacteria shapes the phenotypic and functional profile of intestinal DCs to produce effects that range from their activation and enhanced functions to their exhaustion and tolerance.

Hypoxia inducible factor 1-alpha (HIF-1 alpha) is induced during reperfusion after renal ischemia and is critical for proximal tubule cell survival.

Acute tubular necrosis (ATN) caused by ischemia/reperfusion (I/R) during renal transplantation delays allograft function. Identification of factors that mediate protection and/or epithelium recovery could help to improve graft outcome. We studied the expression, regulation and role of hypoxia inducible factor 1-alpha (HIF-1 α), using in vitro and in vivo experimental models of I/R as well as human post-transplant renal biopsies. We found that HIF-1 α is stabilized in proximal tubule cells during ischemia and unexpectedly in late reperfusion, when oxygen tension is normal. Both inductions lead to gene expression in vitro and in vivo. In vitro interference of HIF-1 α promoted cell death and in vivo interference exacerbated tissue damage and renal dysfunction. In pos-transplant human biopsies, HIF-1 α was expressed only in proximal tubules which exhibited normal renal structure with a significant negative correlation with ATN grade. In summary, using experimental models and human biopsies, we identified a novel HIF-1 α induction during reperfusion with a potential critical role in renal transplant.

Differential resolution of inflammation and recovery after renal ischemia-reperfusion injury in Brown Norway compared with Sprague Dawley rats.

To investigate mechanisms conferring susceptibility or resistance to renal ischemia, we used two rat strains known to exhibit different responses to ischemia-reperfusion. We exposed proximal tubule cells isolated from Sprague Dawley or Brown Norway rats, to a protocol of hypoxia, followed by reoxygenation in vitro. The cells isolated from both rat strains exhibited comparable responses in the disruption of intercellular adhesions and cytoskeletal damage. In vivo, after 24 h of reperfusion, both strains showed similar degrees of injury. However, after 7 days of reperfusion, renal function and tubular structure almost completely recovered and inflammation resolved, but only in Brown Norway rats. Hypoxia-inducible factor-dependent gene expression, ERK1/2, and Akt activation were different in the two strains. Inflammatory mediators MCP-1, IL-10, INF-gamma, IL-1beta, and TNF-alpha were similarly induced at 24 h in both strains but were downregulated earlier in Brown Norway rats, which correlated with shorter NFkappaB activation in the kidney. Moreover, VLA-4 expression in peripheral blood lymphocytes and VCAM-1 expression in kidney tissues were initially similar at 24 h but reached basal levels earlier in Brown Norway rats. The faster resolution of inflammation in Brown Norway rats suggests that this strain might be a useful experimental model to determine the mechanisms that promote repair of renal ischemia-reperfusion injury.