Implications of KRAS mutations in acquired resistance to treatment in NSCLC

Rationale: KRAS is the most common and, simultaneously, the most ambiguous oncogene implicated in human cancer. Despite KRAS mutations were identified in Non Small Cell Lung Cancers (NSCLCs) more than 20 years ago, selective and specific inhibitors aimed at directly abrogating KRAS activity are not yet available. Nevertheless, many therapeutic approaches have been developed potentially useful to treat NSCLC patients mutated for KRAS and refractory to both standard chemotherapy and targeted therapies. The focus of this review will be to provide an overview of the network related to the intricate molecular KRAS pathways, stressing on preclinical and clinical studies that investigate the predictive value of KRAS mutations in NSCLC patients. Materials and Methods: A bibliographic search of the Medline database was conducted for articles published in English, with the keywords KRAS, KRAS mutations in non-small cell lung cancer, KRAS and tumorigenesis, KRAS and TKIs, KRAS and chemotherapy, KRAS and monoclonal antibody, KRAS and immunotherapy, KRAS and drugs, KRAS and drug resistance

Concise Review: Chronic Myeloid Leukemia: Stem Cell Niche and Response to Pharmacologic Treatment

Nowadays, more than 90% of patients affected by chronic myeloid leukemia (CML) survive with a good quality of life, thanks to the clinical efficacy of tyrosine kinase inhibitors (TKIs). Nevertheless, point mutations of the ABL1 pocket occurring during treatment may reduce binding of TKIs, being responsible of about 20% of cases of resistance among CML patients. In addition, the presence of leukemic stem cells (LSCs) represents the most important event in leukemia progression related to TKI resistance. LSCs express stem cell markers, including active efflux pumps and genetic and epigenetic alterations together with deregulated cell signaling pathways involved in self-renewal, such as Wnt/β-catenin, Notch, and Hedgehog. Moreover, the interaction with the bone marrow microenvironment, also known as hematopoietic niche, may influence the phenotype of surrounding cells, which evade mechanisms controlling cell proliferation and are less sensitive or frankly resistant to TKIs. This Review focuses on the role of LSCs and stem cell niche in relation to response to pharmacological treatments. A literature search from PubMed database was performed until April 30, 2017, and it has been analyzed according to keywords such as chronic myeloid leukemia, stem cell, leukemic stem cells, hematopoietic niche, tyrosine kinase inhibitors, and drug resistance. Stem Cells Translational Medicine 2018

CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation

Identification of a targetable KRAS-mutant epithelial population in non-small cell lung cancer

Lung cancer is the leading cause of cancer deaths. Tumor heterogeneity, which hampers development of targeted therapies, was herein deconvoluted via single cell RNA sequencingin aggressive human adenocarcinomas (carrying Kras-mutations) and comparable murine model. We identified a tumor-specific, mutant-KRAS-associated subpopulation which is conserved in both human and murine lung cancer. We previously reported a key role for the oncogene BMI-1 in adenocarcinomas. We therefore investigated the effects of in vivo PTC596 treatment, which affects BMI-1 activity, in our murine model. Post-treatment, MRI analysis showed decreased tumor size, while single cell transcriptomics concomitantly detected near complete ablation of the mutant-KRAS-associated subpopulation, signifying the presence of a pharmacologically targetable, tumor-associated subpopulation. Our findings therefore hold promise for the development of a targeted therapy for KRAS-mutant adenocarcinomas

CDK4/6 Inhibitors Overcome Endocrine ESR1 Mutation-Related Resistance in Metastatic Breast Cancer Patients

ESR1 mutations contribute to endocrine resistance and occur in a high percentage of hormone-receptor-positive (HR+) metastatic breast cancer (mBC) cases. Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) changed the treatment landscape of HR+ mBC, as they are able to overcome estrogen resistance. The present retrospective study investigates the clinical benefit of CDK4/6i in ESR1 mutant HR+ mBC patients treated with a CDK4/6i as first- or second-line therapy. Plasma was collected at baseline prior to CDK4/6i plus hormone therapy as a first- or second-line treatment. Circulating free DNA (cfDNA) was extracted from plasma, and ESR1 mutation analysis was performed on a ddPCR. Statistical analyses were performed to investigate the predictive power of ESR1 mutations and any association with clinical factors. A total of 42 patients with mBC treated with CDK4/6i plus endocrine therapy as first- (n = 35) or second-line (n = 7) were enrolled. Twenty-eight patients received hormonal therapy (AI or tamoxifen) in the adjuvant setting. ESR1 mutation status in blood was associated with shorter median disease-free survival (DFS) (30 vs. 110 months; p = 0.006). Multivariate analysis confirmed ESR1 mutations as independent factors of resistance in adjuvant hormone therapy. On the contrary, no difference in progression-free survival (PFS) was observed in the presence or absence of an ESR1 mutation in patients treated with CDK4/6i as first-line treatment (p = 0.29). No statistically significant correlation between the best response to CDK4/6i and ESR1 mutation was found (p = 0.46). This study indicates that the ESR1 mutation detected in cfDNA is an independent predictive factor of clinical recurrence in the adjuvant setting and that CDK4/6i can overcome ESR1-dependent resistance

Analysis of biomarkers in plasma-derived exosomal RNA in prostate cancer and application in combinatorial treatments targeting signal transduction of androgen receptor

The first part of the study was aimed to investigate the expression levels of panels of small RNA markers for PCa using plasma samples from men with various stages of disease. Plasma samples from healthy donor and cancer patients were used to establish the best small RNA isolation method and qPCR efficiency and to set up and optimize a technique for the detection of miRNA, snoRNA, sdRNA and tRFs and evaluate their diagnostic prognostic, predictive, and monitoring potential. Expression analysis were done by Real-Time PCR of RNA extracted from 200 µL of plasma. The study demonstrates the feasibility of small RNA isolation method in plasma samples and detection by qPCR. The evaluation of the diagnostic and prognostic role of these miRNA on plasma samples from cancer patients should be confirmed. The second part of the study was aimed to investigate the role of AR-V7 and AR-FL to predict resistance to hormonal therapy (HT) in castration resistant prostate cancer (CRPC) and to develop a new methodological approach based on plasma-derived exosomal RNA. Blood samples were collected from 88 CRPC patients before first or second-line HT. RNA was isolated from extracellular vesicles and analysed for AR-FL and AR-V7 by ddPCR. AR-FL was detected in all patients, whereas 29% of these patients were AR-V7+ before starting hormonal therapy. There was a significant higher expression of AR-FL mRNA in AR-V7+ compared to AR-V7- patients (p<0.0001). Median PFS and OS were significantly longer in AR-V7- vs AR-V7+ patients (median PFS 25 vs 4 months, p<0.0001; median OS 9 vs 38 months, p<0.0001). The study suggests that resistance to ADT is associated to AR-V7 status, and that AR-FL expression may also identify a category of patients borderline for their response to HT. The third part of the study was aimed to evaluate the combined treatment of abiraterone (anti-AR pathway) and everolimus (mTOR inhibitor) in prostate cancer cell lines expressing the AR-V7. The combined treatment induced a significant increase of concentration-dependent cytotoxicity and apoptosis compared to single treatment strategy

La farmacogenetica nella medicina di precisione in oncologia: applicazioni nella pratica clinica e nuove prospettive

La farmacogenetica è un ramo della farmacologia clinica che studia le variazioni genetiche e i loro effetti sulla risposta individuale alle terapie. Questa disciplina cerca di comprendere come le differenze nel patrimonio genetico di una persona influenzino l'efficacia e la sicurezza dei farmaci. La farmacogenetica trova principalmente applicazione nelle terapie oncologiche, sia di tipo chemioterapico che a bersaglio molecolare, valutando le correlazioni con le tossicità o fenomeni di resistenza e/o sensibilità farmacologiche. Nell’ottica di una medicina personalizzata, la comprensione del ruolo funzionale e dell’impatto clinico di polimorfismi genetici può consentire ai clinici di personalizzare il trattamento terapeutico e prevenire fenomeni di sensibilità, resistenza, esposizione a effetti avversi importanti e tossicità farmacologica. In questa tesi è stata approfondita l’analisi di polimorfismi a carico dei geni DPYD, UGT1A1, ENOS, CYP17A1, ABCB1 e CDA e la loro correlazione con la presenza di eventi avversi o l’efficacia di chemio o ormonoterapie. Inoltre, è stata valutata l’applicabilità della biopsia liquida e il ruolo di potenziali biomarcatori in diversi tumori solidi per la risposta o la resistenza a terapie a bersaglio molecolare (i.e. CDK4/6i, EGFR-TKIs, anti-PD1). Sono stati prelevati campioni di sangue da pazienti affetti da tumori solidi ed il DNA germinale o gli acidi nucleici circolanti (DNA, RNA e microvescicole) sono stati isolati ed analizzati tramite Real-Time PCR, digital PCR o Next-Genaration Sequencing (NGS). Le analisi hanno rilevato possibili associazioni tra i polimorfismi a carico dei geni DPYD, UGT1A1, ENOS, CYP17A1, ABCB1 e CDA e la tossicità o la resistenza alla terapia. La biopsia liquida si è confermata un abile strumento utile per la valutazione di potenziali biomarcatori di risposta o resistenza a terapie target. Un approccio multi-parametrico e l’inserimento di tali analisi in protocolli di studio permetterà una migliore personalizzazione della terapia e sarà di supporto nel management clinico dei pazienti affetti da tumori solidi

Metronomic Chemotherapy in Pediatric Oncology: From Preclinical Evidence to Clinical Studies

Metronomic chemotherapy (MC) is the frequent, regular administration of drug doses designed to maintain a low, but active, range of concentrations of chemotherapeutic drugs, during prolonged periods of time without inducing excessive toxicities. To date, more than 400,000 children and adolescents under the age of 20 are diagnosed with cancer, per year, with 80% survival in most high-income countries, but less than 30% in low- and middle-income ones. In this review, we summarized the principal preclinical and clinical studies involving the use of MC in the most common pediatric tumors, with an overview of efficacy, toxicity, pharmacokinetic profile, and biomarkers. The best advantages of MC are low toxicity, oral administration and, thus, the feasibility of a more comfortable, home-based treatment, therefore improving the quality of life of the children themselves and of their parents and caregivers. Moreover, MC could represent a valid method to reduce the economic burden of anticancer therapy in the pediatric setting

Gene Expression Profile of Colon Mucosa after Cytotoxic Insult in wt and Apc-Mutated Pirc Rats: Possible Relation to Resistance to Apoptosis during Carcinogenesis

Apc-mutated Pirc rats, spontaneously developing intestinal tumours, are resistant to 1,2-dimethylhydrazine- (DMH-) induced colon apoptosis. To understand this phenomenon, we analyzed the expression of genotoxic stress-related genes Mgmt, Gsta1, and Gstp1 in the colon of wt and Pirc rats in basal conditions and 24 h after DMH; plasmatic oxidant/antioxidant status was also evaluated. After DMH, Mgmt expression was increased in both genotypes but significantly only in wt rats; Gsta1 expression was significantly increased in both genotypes. Gstp1 expression did not vary after DMH but was lower in Pirc rats. Moreover, for each genotype, we studied by microarray technique whole gene expression profile after DMH. By unsupervised cluster analysis, 28 genes were differentially modulated between the two genotypes. Among them were interferon-induced genes Irf7, Oas1a, Oasl2, and Isg15 and the transcription factor Taf6l, overexpressed in DMH-treated wt rats and unchanged in Pirc rats. RT-PCR confirmed their overexpression in DMH-treated wt rats and showed a slighter variation in DMH-treated Pirc rats. Taken together, despite a blunted induction of Irf7, Oas1a, and Mgmt, defective apoptosis in Pirc rats 24 h after DMH is not mirrored by major differences in gene expression compared with wt rats

KRAS mutations as potential mechanism of crizotinib acquired resistance: a study on circulating tumor DNA.

Background: Crizotinib is an effective treatment for patients with ALK rearranged NSCLC but, unfortunately, patients progress with a median of 9-10 months. Resistance could be acquired through ALK point mutations, amplification or activation of alternative pathways i.e. KIT, EGFR and KRAS [1]. Second-generation TKIs demonstrated an enhanced activity in crizotinib-resistant patients that retain ALK addiction. However, rebiopsy represents a critical issue in lung cancer and analysis of circulating tumor DNA (cftDNA) has a promising role for the identification of resistance to treatment. Methods: Sixteen NSCLC patients with ALK rearrangement were enrolled after progression to ALK-TKI. Blood was collected at several time points during and after TKI treatment. CftDNA was extracted from plasma using QIAamp circulating nucleic acid kit (Qiagen) and ALK and KRAS codon 12 mutations were tested using the Digital Droplet PCR (ddPCR - BioRad). Results: All patients were stage IV adenocarcinoma; 15 patients received crizotinib and 1 ceritinib. ALK-TKIs were administered mainly as second-line, in 2 cases as first and in the remaining as third-line therapy. The best responses to treatment were 12 partial responses (PRs), 3 stable diseases (SDs) and 1 progressive disease (PD). The median PFS was 8 months. ALK point mutations alone were identified in 2 patients; one showed both p.L1196M and p.G1269A mutations. Six patients were carriers of a KRAS mutation at crizotinib PD, 2 of them presented both ALK and KRAS mutations. CftDNA was monitored during second generation ALK-TKI therapy and the amount of both ALK or KRAS mutations decreased along with tumor response. Interestingly, one additional extra-patient analysed before treatment with crizotinib was carrying the ALK p.F1174L mutation and showed primary refractoriness. Conclusions: KRAS and ALK mutations induce acquired resistance to TKIs targeting ALK rearrangement. The detection of KRAS mutation in circulating tumor DNA could represent a predictive marker of acquired resistance. Moreover, the quantification of mutant alleleS burden could be useful to monitor treatment response. 1. Katayama R, et al. Sci Transl Med. 2012 Feb;4(120):120ra1