A phase Ib/II trial of capmatinib plus spartalizumab vs. spartalizumab alone in patients with pretreated hepatocellular carcinoma.

Background & aims: This phase Ib/II trial evaluated the safety and efficacy of capmatinib in combination with spartalizumab or spartalizumab alone in patients with advanced hepatocellular carcinoma (HCC). Methods: Eligible patients who had progressed or were intolerant to sorafenib received escalating doses of capmatinib 200 mg, 300 mg, and 400 mg twice a day (bid) plus spartalizumab 300 mg every 3 weeks (q3w) in the phase Ib study. Once the recommended phase II dose (RP2D) was determined, the phase II study commenced with randomised 1:1 treatment with either capmatinib + spartalizumab (n = 32) or spartalizumab alone (n = 30). Primary endpoints were safety and tolerability (phase Ib) and investigator-assessed overall response rate per RECIST v1.1 for combination vs. single-agent arms using a Bayesian logistic regression model (phase II). Results: In phase Ib, the RP2D for capmatinib in combination with spartalizumab was determined to be 400 mg bid. Dose-limiting toxicity consisting of grade 3 diarrhoea was reported in one patient at the capmatinib 400 mg bid + spartalizumab 300 mg q3w dose level. The primary endpoint in the phase II study was not met. The observed overall response rate in the capmatinib + spartalizumab arm was 9.4% vs. 10% in the spartalizumab arm. The most common any-grade treatment-related adverse events (TRAEs, ≥20%) were nausea (37.5%), asthenia and vomiting (28.1% each), diarrhoea, pyrexia, and decreased appetite (25.0% each) in the combination arm; TRAEs ≥10% were pruritus (23.3%), and rash (10.0%) in the spartalizumab-alone arm. Conclusion: Capmatinib at 400 mg bid plus spartalizumab 300 mg q3w was established as the RP2D, with manageable toxicities and no significant safety signals, but the combination did not show superior clinical activity compared with spartalizumab single-agent treatment in patients with advanced HCC who had previously been treated with sorafenib. Impact and implications: Simultaneous targeting of MET and programmed cell death protein 1 may provide synergistic clinical benefit in patients with advanced HCC. This is the first trial to report a combination of capmatinib (MET inhibitor) and spartalizumab (programmed cell death protein 1 inhibitor) as second-line treatment after sorafenib for advanced HCC. The combination did not show superior clinical activity compared with spartalizumab single-agent treatment in patients with advanced HCC who had previously been treated with sorafenib. The results indicate that there is a clear need to identify a reliable predictive marker of response for HCC and to identify patients with HCC that would benefit from the combination of checkpoint inhibitor +/- targeted therapy. Clinical trial number: NCT02795429.

FOXM1 Transcription Factor: A New Component of Chronic Myeloid Leukemia Stem Cell Proliferation Advantage.

FOXM1 transcription factor is a central component of tumor initiation, growth, and progression due to its multiple effects on cell cycle, DNA repair, angiogenesis and invasion, chromatin, protein anabolism, and cell adhesion. Moreover, FOXM1 interacts with ß-catenin promoting its nuclear import and transcriptional activation. Here, we show that FOXM1 is involved in the advantage of chronic myeloid leukemia hematopoiesis over the normal counterpart. FOXM1 hyper-activation associated with BCR-ABL1 results from phosphorylation by the fusion protein kinase-dependent activation of Polo-like kinase 1. FOXM1 phosphorylation lets its binding with ß-catenin and ß-catenin transcriptional activation, a key event for persistence of the leukemic stem cell compartment under tyrosine kinase inhibitor therapy. Polo-like kinase 1 inhibitor BI6727, already advanced for clinical use, breaks ß-catenin interaction with FOXM1, hence hampering FOXM1 phosphorylation, ß-catenin binding, nuclear import, and downstream signaling. In conclusion, our results support Polo-like kinase 1/FOXM1 axis as a complementary target to eradicate leukemic early progenitor/stem cell compartment in chronic myeloid leukemia. J. Cell. Biochem. 118: 3968-3975, 2017. © 2017 Wiley Periodicals, Inc.

14-3-3 Binding and Sumoylation Concur to the Down-Modulation of ß-catenin Antagonist chibby 1 in Chronic Myeloid Leukemia.

The down-modulation of the ß-catenin antagonist Chibby 1 (CBY1) associated with the BCR-ABL1 fusion gene of chronic myeloid leukemia (CML) contributes to the aberrant activation of ß-catenin, particularly in leukemic stem cells (LSC) resistant to tyrosine kinase (TK) inhibitors. It is, at least partly, driven by transcriptional events and gene promoter hyper-methylation. Here we demonstrate that it also arises from reduced protein stability upon binding to 14-3-3σ adapter protein. CBY1/14-3-3σ interaction in BCR-ABL1+ cells is mediated by the fusion protein TK and AKT phosphorylation of CBY1 at critical serine 20, and encompasses the 14-3-3σ binding modes I and II involved in the binding with client proteins. Moreover, it is impaired by c-Jun N-terminal kinase (JNK) phosphorylation of 14-3-3σ at serine 186, which promotes dissociation of client proteins. The ubiquitin proteasome system UPS participates in reducing stability of CBY1 bound with 14-3-3σ through enhanced SUMOylation. Our results open new routes towards the research on molecular pathways promoting the proliferative advantage of leukemic hematopoiesis over the normal counterpart.

DNA methyltransferase 1 drives transcriptional down-modulation of ß catenin antagonist Chibby1 associated with the BCR-ABL1 gene of chronic myeloid leukemia.

The decrease of Chibby1 (CBY1) contributes to ß catenin constitutive activation associated with the presence of the BCR-ABL1 fusion gene of chronic myeloid leukemia (CML). This is mediated by transcriptional events and driven by DNA hyper-methylation at promoter-associated CpG islands of the CBY1-encoding gene C22orf2. Moreover, CBY1 transcriptional induction proceeding from promoter de-methylation is a component of BCR-ABL1+ cell response to Imatinib (IM). Our study showed that DNA methyltransferase 1 (DNMT1) has a central role in the hyper-methylation at the C22orf2 promoter. Further investigation in leukemic hematopoietic progenitors from IM-responsive and IM-resistant CML patients at diagnosis failed to demonstrate any correlation between DNMT1-driven hyper-methylation of the C22orf2 promoter and response to IM. Notably, the response to IM was neither predicted by DNMT1-driven hyper-methylation of BCL2-like11 at diagnosis. In conclusion, the hypermethylation of C22orf2 and BCL2-like11 promoters proceeding from DNMT1 is a crucial component of their reduced expression, but it is not directly involved in CML resistance to IM. It might rather contribute to the disease evolution towards a drug-resistant phenotype in more advanced phases or blast crisis.

A calpain-cleaved fragment of ß-catenin promotes BCRABL1+ cell survival evoked by autophagy induction in response to imatinib.

Autophagy protects chronic myeloid leukemia stem cells from tyrosine kinase inhibitors hence supporting the disease persistence under therapy. However, the signals involved in autophagy regulation relative to BCR-ABL1 are still elusive. The autophagic flux proceeding from the inhibition of BCR-ABL1 tyrosine kinase represents a regulatory mechanism of ß-catenin stability through events encompassing the activation of calpain, which targets ß-catenin for proteasome-independent degradation. Accordingly, its inactivation may contribute to induce autophagy and autophagy induction may, in turn, promote ß-catenin autolysosomal degradation to originate a regulatory loop where ß-catenin plays a central role in cell decision between life and death. Here we proved that the cytoplasmic accumulation of ß-catenin driven by up-regulation of its antagonist Chibby1 is a component of autophagy induction in response to imatinib in BCR-ABL1+ cells opposing the apoptotic death. It is contingent upon ER stress and elevation of free Ca(2+) cytosolic concentration and results in the calpain cleavage into a 28kDa fragment implicated in ß-catenin proteasome-independent degradation. More important for BCR-ABL1+ cell survival and proliferation following IM treatment, might be the calpain-mediated cleavage of ß-catenin accumulated within the cytoplasmic compartment into a 75kDa fragment, still owning TCF-dependent transcriptional activity. Such a ß-catenin fragment might be crucial for BCR-ABL1+ cell survival following the fusion protein TK inhibition.

BCR-ABL1-associated reduction of beta catenin antagonist Chibby1 in chronic myeloid leukemia.

Beta Catenin signaling is critical for the self-renewal of leukemic stem cells in chronic myeloid leukemia. It is driven by multiple events, enhancing beta catenin stability and promoting its transcriptional co-activating function. We investigated the impact of BCR-ABL1 on Chibby1, a beta catenin antagonist involved in cell differentiation and transformation. Relative proximity of the Chibby1 encoding gene (C22orf2) on chromosome 22q12 to the BCR breakpoint (22q11) lets assume its involvement in beta catenin activation in chronic myeloid leukemia as a consequence of deletions of distal BCR sequences encompassing one C22orf2 allele. Forty patients with chronic myeloid leukemia in chronic phase were analyzed for C22orf2 relocation and Chibby1 expression. Fluorescent in situ hybridization analyses established that the entire C22orf2 follows BCR regardless of chromosomes involved in the translocation. In differentiated hematopoietic progenitors (bone marrow mononuclear cell fractions) of 30/40 patients, the expression of Chibby1 protein was reduced below 50% of the reference value (peripheral blood mononuclear cell fractions of healthy persons). In such cell context, Chibby1 protein reduction is not dependent on C22orf2 transcriptional downmodulation; however, it is strictly dependent upon BCR-ABL1 expression because it was not observed at the moment of major molecular response under tyrosine kinase inhibitor therapy. Moreover, it was not correlated with the disease prognosis or response to therapy. Most importantly, a remarkable Chibby1 reduction was apparent in a putative BCR-ABL1+ leukemic stem cell compartment identified by a CD34+ phenotype compared to more differentiated hematopoietic progenitors. In CD34+ cells, Chibby1 reduction arises from transcriptional events and is driven by C22orf2 promoter hypermethylation. These results advance low Chibby1 expression associated with BCR-ABL1 as a component of beta catenin signaling in leukemic stem cells.

Chibby drives ß catenin cytoplasmic accumulation leading to activation of the unfolded protein response in BCR-ABL1+ cells.

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL fusion protein. However, the phenotype of leukemic stem cells (LSC) is sustained by ß catenin rather than by the BCR-ABL TK. ß catenin activity in CML is contingent upon its stabilization proceeding from the BCR-ABL-induced phosphorylation at critical residues for interaction with the Adenomatous polyposis coli (APC)/Axin/glycogen synthase kinase 3 (GSK3) destruction complex or GSK3 inactivating mutations. Here we studied the impact of ß catenin antagonist Chibby (CBY) on ß catenin signaling in BCR-ABL1+ cells. CBY is a small conserved protein which interacts with ß catenin and impairs ß catenin-mediated transcriptional activation through two distinct molecular mechanisms: 1) competition with T cell factor (TCF) or lymphoid enhancer factor (LEF) for ß catenin binding; and 2) nuclear export of ß catenin via interaction with 14-3-3. We found that its enforced expression in K562 cell line promoted ß catenin cytoplasmic translocation resulting in inhibition of target gene transcription. Moreover, cytoplasmic accumulation of ß catenin activated the endoplasmic reticulum (ER) stress-associated pathway known as unfolded protein response (UPR). CBY-driven cytoplasmic accumulation of ß catenin is also a component of BCR-ABL1+ cell response to the TK inhibitor Imatinib (IM). It evoked the UPR activation leading to the induction of BCL2-interacting mediator of cell death (BIM) by UPR sensors. BIM, in turn, contributed to the execution phase of apoptosis in the activation of ER resident caspase 12 and mobilization of Ca(2+) stores.

Cytoplasmatic compartmentalization by Bcr-Abl promotes TET2 loss-of-function in chronic myeloid leukemia.

The loss-of-function of ten-eleven-translocation (TET) 2, a Fe(2+) -oxoglutarate-dependent dioxygenase catalyzing 5 methyl cytosine (5mC) conversion into 5-hydroxymethylcytosine (5hmC), contributes to the hematopoietic transformation in vivo. The aim of our study was to elucidate its role in the phenotype of chronic myeloid leukemia (CML), a myeloproliferative disease caused by the Bcr-Abl rearranged gene. We first confirmed TET2 interaction with the Bcr-Abl protein predicted by a Fourier-based bioinformatic method. Such interaction led to TET2 cytoplasmatic compartmentalization in a complex tethered by the fusion protein tyrosine kinase (TK) and encompassing the Forkhead box O3a (FoxO3a) transcription factor. We then focused the impact of TET2 loss-of-function on epigenetic transcriptional regulation of Bcl2-interacting mediator (BIM), a pro-apoptotic protein transcriptionally regulated by FoxO3a. BIM downregulation is a critical component of CML progenitor extended survival and is also involved in the disease resistance to imatinib (IM). Here we reported that TET2 release from Bcr-Abl protein following TK inhibition in response to IM triggers a chain of events including TET2 nuclear translocation, re-activation of its enzymatic function at 5mC and recruitment at the BIM promoter followed by BIM transcriptional induction. 5hmC increment following TET2 re-activation was associated with the reduction of histone H3 tri-methylation at lysine 9 (H3K9me3), which may contribute with DNA de-methylation reported elsewhere to recast a permissive epigenetic "landscape" for FoxO3a transcriptional activity.

Gadd45a transcriptional induction elicited by the Aurora kinase inhibitor MK-0457 in Bcr-Abl-expressing cells is driven by Oct-1 transcription factor.

The advantage of Aurora kinase (AK) inhibitors in chronic myeloid leukemia (CML) therapy mostly arises from "off-target" effects on tyrosine kinase (TK) activity of wild type (wt) or mutated Bcr-Abl proteins which drive the disease resistance to imatinib (IM). We proved that the AK inhibitor MK-0457 induces the growth arrest DNA damage-inducible (Gadd) 45a through recruitment of octamer-binding (Oct)-1 transcription factor at a critical promoter region for gene transcription and covalent modifications of histone H3 (lysine 14 acetylation, lysine 9 de-methylation). Such epigenetic chromatin modifications may depict a general mechanism promoting the re-activation of tumor suppressor genes silenced by Bcr-Abl.

Comparison of Abbott RealTime High Risk HPV and Hybrid Capture 2 for the detection of high-risk HPV DNA in a referral population setting.

BACKGROUND: The Abbott RealTime High Risk HPV assay (ART) is an automated multiplex real-time PCR test for detection of DNA from 14 high risk (HR) HPV types in cervical specimens and simultaneous distinction of HPV16 and HPV18 from other HR-HPV. OBJECTIVES: To evaluate the performance of the ART assay in specimens referred for HPV testing to our laboratory (referral population) by comparison with historical data from HC2 and INNO-LiPA as well as histological status, if available. STUDY DESIGN: 412 cervical specimens were collected from women between 18 and 70 years of age: 301 previously tested by HC2 without clinical data and 111 previously tested by HC2 and INNO-LiPA with histological diagnosis of CIN3+. RESULTS: Our study demonstrated good overall agreement between ART, HC2 and INNO-LiPA. In the group of the CIN3+ specimens HR-HPV was detected by ART in 93.07% (95% CI: 88.12-98.02), while HR-HPV detection rates with HC2 and INNO-LiPA were 91.09% (95% CI: 85.53-96.65) and 95.05% (95% CI: 90.82-99.28), respectively. The typing capability of ART for HPV16, HPV18 and a pool of twelve other HR-HPV types was investigated by comparison with INNO-LiPA demonstrating high overall assay concordance (89.81%; k 0.87). CONCLUSIONS: The Abbott RealTime assay showed similar clinical performance for detection of CIN3+ compared with HC2. The high level of automation and ability to identify HPV16, HPV18 and other HR-HPV make this assay a very attractive option for HR-HPV testing, potentially improving patient management by risk stratification of cytological abnormal populations.

Molecular analysis of HPV 16 E6I/E6II spliced mRNAs and correlation with the viral physical state and the grade of the cervical lesion.

The presence of HPV 16 E6*I/E6*II spliced transcripts, in cervical lesions of different grade, was analyzed to characterize the transcription pattern. The presence and amount of spliced transcripts were correlated with DNA viral markers such as E2/E6 ratio and physical state. The detection of HPV 16 E6*I/E6*II mRNAs was set up by an SYBR Green real-time reverse transcriptase PCR assay with an optimal dynamic range and sensitivity. The assay was applied to the analysis of 71 specimens, positive to HPV 16 as a sole infection, from women with abnormal cervical smears, precisely 31 low-grade squamous intraepithelial lesions and 40 high-grade lesions. Samples negative to both transcripts were found only in low-grade cervical lesions. Three different transcription profiles were found in the low- and high-grade lesions analyzed: in low-grade lesions samples positive only to E6*II and in high-grade lesions samples positive only to E6*I were detected. In low- and high-grade lesions, samples positive to both E6*I and E6*II were found. In the samples positive for both transcripts, the E6*I/E6*II ratio was higher than that in the majority of high-grade lesions and lower than that in all the low-grade lesions. Analyzing the transcription pattern in relation to E2/E6 ratio and to the DNA physical state, the presence of high values of E6*I was associated mainly with low values of E2/E6 ratio and of mixed DNA forms. The detection of HPV 16 E6*I/E6*II mRNAs may serve to identify transcription patterns indicative of cervical disease progression and help physicians to decide clinical management.

High-throughput two-step LNA real time PCR assay for the quantitative detection and genotyping of HPV prognostic-risk groups.

BACKGROUND: Human papillomavirus (HPV) infection is a necessary event in the development of cervical carcinoma. High risk (HR) HPV genotypes, however, may progress differentially from low grade lesions to malignancy. OBJECTIVES: The necessity to genotype and quantify HPV-DNA in cervical screening programs, in the follow up post-surgical treatments and in monitoring the effectiveness of HPV vaccination programs, requires access to economical, high-throughput and flexible molecular technologies. STUDY DESIGN: A high-throughput two-step LNA real time PCR assay was developed consisting of real time PCR reactions with fluorescent Locked Nucleic Acid (LNA) probes. The first step permits classification into three prognostic-risk groups of nine HR HPV genotypes (16, 18, 31, 33, 35, 45, 52, 56 and 58) most frequently found associated with cervical lesions in Europe. The second step allows us to genotype/quantify the HPV-DNA only when clinical, epidemiological or prophylactic aims exist. RESULTS: The specificity, repeatability, detection and quantitation limit, and linearity of the assay were evaluated and appear to be in agreement with guidelines for the validation of analytical procedures. The overall genotype concordance on cervical samples between our assay and INNOLiPA test was 94% (k 0.83) indicating good agreement. CONCLUSIONS: The two-step PCR assay can give much information relative to the predictive value of different HR HPV types and can quantify the genotype-specific viral load. In particular, its ability to detect and quantify nine HR HPV genotypes can help provide more efficient and successful patient care and may be useful for the monitoring of the efficacy of HPV vaccines.

Disruption of HPV 16 E1 and E2 genes in precancerous cervical lesions.

The presence of HPV 16 E1 and E2 genes was detected in cervical cytological samples using polymerase chain reaction assays. A total of 48 samples were analyzed from patients with HPV 16 infections associated with 13 low-grade cervical intraepithelial neoplasia and 35 high-grade cervical intraepithelial neoplasia. Disruption/deletion sites, within E1 and E2 genes, were detected using 6 primer pairs spanning the entire gene sequences. This technique is not able to recognize mixed DNA forms (integrated plus episomal DNA); therefore, it detects only the presence of pure integrated DNA. Both E1 and E2 genes were detected in 84.6% and in 62.9% of low and high-grade lesions, respectively. The rate of samples with disrupted/deleted genes was significantly higher in high-grade cervical intraepithelial neoplasia than in low-grade cervical intraepithelial neoplasia (P<0.05). In high-grade cervical intraepithelial neoplasia the disruption/deletion pattern involved both E1 and E2 genes and E2 gene was always involved, while in the low grade cervical intraepithelial neoplasia only E1 gene was involved. In conclusion, in high-grade cervical lesions E2 gene seems a suitable target to identify HPV 16 DNA integration into cellular genome.

Correlation of high-risk human papillomavirus genotypes persistence and risk of residual or recurrent cervical disease after surgical treatment.

The evidence on genotype-specific risk in women infected with human papillomavirus (HPV) with normal cytology and the importance of the distinction of high-risk (HR)-HPV genotypes in the management of low-grade lesions suggest that the distinction of HR-HPV genotypes has the potential to improve the follow-up of patients treated for high-grade cervical lesions. The aims of this study were to define the persistence of the different HR-HPV in the follow-up of surgical treated women, to detect the changes of genotypes from the pre- to the post-operative status, and to evaluate whether genotype-specific persistence can predict the development of residual or recurrent disease during the follow-up. HR-HPV detection and genotyping was carried out by the Linear Array HPV Genotyping Test on cervical cytological samples from 72 women treated by surgery. The 6-month post-operative HPV status was correlated with the pre-operative HPV genotype and with the residual or recurrent disease within 24 months. It was observed that the residual or recurrent disease in women with persistence of HPV 16 and/or HPV 18 was higher (82.4%) than in women with persistence of at least one HR-HPV type of group 2 (HPV 31, 33, 35, 45, 52, and 58) (66.7%) and at least one type of group 3 (HPV 39, 51, 56, 59, 68, 26, 53, 66, 73, and 82) (14.3%). These data defined HR-HPV groups for the risk of progression of disease and suggested that the identification of persistent infection with different HR-HPV genotypes has the potential to improve the management of these patients.

SRP-27 is a novel component of the supramolecular signalling complex involved in skeletal muscle excitation-contraction coupling.

SRP-27 (sarcoplasmic reticulum protein of 27 kDa) is a newly identified integral membrane protein constituent of the skeletal muscle SR (sarcoplasmic reticulum). We identified its primary structure from cDNA clones isolated from a mouse skeletal muscle cDNA library. ESTs (expressed sequence tags) of SRP-27 were found mainly in cDNA libraries from excitable tissues of mouse. Western blot analysis confirmed the expression of SRP-27 in skeletal muscle and, to a lower extent, in heart and brain. Mild trypsin proteolysis combined with primary-structure prediction analysis suggested that SRP-27 has four transmembrane-spanning alpha helices and its C-terminal domain faces the cytoplasmic side of the endo(sarco)plasmic reticulum. The expression of SRP-27 is higher in fast twitch skeletal muscles compared to slow twitch muscles and peaks during the first month of post-natal development. High-resolution immunohistochemistry and Western blot analysis of subcellular fractions indicated that SRP-27 is distributed in both longitudinal tubules and terminal cisternae of the SR, as well as in the perinuclear membrane systems and the nuclear envelope of myotubes and adult fibres. SRP-27 co-sediments with the RyR (ryanodine receptor) macromolecular complex in high-salt sucrose-gradient centrifugation, and is pulled-down by anti-RyR as well as by maurocalcin, a well characterized RyR modulator. Our results indicate that SRP-27 is part of a SR supramolecular complex, suggesting the involvement of SRP-27 in the structural organization or function of the molecular machinery underlying excitation-contraction coupling.