Non-mutated Cells Research Articles

Modelling Single Cell B-Cell Receptor Signaling Reveals Enhanced Activity in Primary CLL Cells Compared to Non-Malignant Cells While Fundamental Network Circuit Topology Remains Stable Even with Novel Therapeutic Inhibitors

B-cell receptor (BCR) signalling is central for the pathomechanism of chronic lymphocytic leukemia (CLL). Novel inhibitors of BCR signalling have recently substantially improved treatment of CLL, and a better characterization of the molecular circuitry of leukemic BCR signalling will allow a more refined targeting of this Achilles heel. In order to model malignant and non-malignant BCR signalling, we quantified after stimulation 5 components of BCR signaling (ZAP70/SYK, BTK, PLCy2, AKT, ERK1/2) in single cells from primary human leukemic and non-malignant tissue via phospho-specific flow cytometry over 6 time points. We stimulated cells from 11 patients and non-malignant CD19 negative enriched B-cells from 5 healthy donors by crosslinking the BCR with anti-IgM and/or anti-CD19 and synchronous inhibition of phosphatases with H2O2. As expected, we found more phosphorylation of all BCR signalling components after stimulation in malignant vs non-malignant cells and in IGHV non-mutated CLL cells compared to IGHV mutated CLL cells. Intriguingly, inhibition of phosphatases with H2O2 led to higher phosphorylation of BCR components in CLL cells with mutated IGHV genes compared to CLL cells with non-mutated IGHV genes, suggesting a stronger dampening of signalling activity in mutated IGHV CLL by phosphatases. In order to characterize the signalling circuitry, we modelled the connectivity of the cascade components by correlating signal intensities across single cells of the cell populations of single samples (Figure 1). Surprisingly, upon stimulation no substantial differences in network topology were observed between malignant and non-malignant cells. To additionally test for changes in network topology, we challenged the BCR signaling cascade with inhibitors for BTK (ibrutinib), PI3K (idelalisib). Ibrutinib and idelalisib acted complementary, but not synergistic, and were similarly effective in IGHV mutated and non-mutated CLL. Effects of idelalisib were the same on malignant and non-malignant cells, whereas ibrutinib was mostly active on CLL cells, not on non-malignant B-cells. Upon stimulation with combinations of IgM and CD19 crosslinking augmented with H2O2, phosphorylation of PLCy2 could not be significantly inhibited by idelalisib or ibrutinib on a timescale of 28mins. We therefore aimed to identify central activating nodes of the BCR signalling cascade using targeted inhibitors. In fact, we found that inhibition of LYN with dasatinib and inhibition of SYK with entospletinib could substantially reduce phosphorylation of PLCy2, BTK and ERK but not AKT after all combinations of BCR stimulation. This suggests additional signalling cascades modulating AKT and a strong impact of SYK/LYN activity on the regulation of PLCy2. In summary, our findings underline the importance of single cell analysis of the dynamic circuitry of B-cell receptor signalling to understand development of resistance mechanisms and potential vulnerabilities. Figure 1: Workflow scheme of the Bayesian network learning and averaging approach. After discretizing the continuous single cell data, an optimal network is derived from each of R bootstrap samples. The Bayesian network learning strategy uses the BDe scoring function and a greedy hill-climbing algorithm to find the network model that represents the resampled data best. An average arc strength for each connection between nodes is derived from the number of occurrences of the respective connection in the set of R best scoring networks. Further averaging among networks derived from different data sets was applied for identifying conditional, temporal, and group-specific differences. Figure 1 Disclosures Döhner: Novartis: Consultancy, Honoraria, Research Funding; Astex: Consultancy, Honoraria; Bristol Myers Swuibb: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Arog: Research Funding; Seattle Genetics: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Pfizer: Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria. Stilgenbauer:GSK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Other: Travel support; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Hoffmann La-Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau.

A Comprehensive Analysis of Rhoa Mutation Positive and Negative Angioimmunoblastic T-Cell Lymphomas By Targeted Deep Sequencing, Expression Profiling, and Single Cell Digital Image Analysis

Angioimmunoblastic T-cell lymphoma is a uniquely aggressive mature T-cell neoplasm. In recent years, recurrent genetic mutations in RHOA, TET2, DNMT3A and IDH2 have been identified which characterize this disease. However, a deep molecular study assessing both DNA mutations and RNA expression profile, combined with digital image analysis is lacking. We designed a study to assess the significance of molecular and morphologic features by high resolution digital image analysis in cases of AITL. Eighteen separate tissues from ten patients with AITL were identified and analyzed in this study. Our results confirm recurrent mutations in RHOA, TET2, DNMT3A, and IDH2, and demonstrate increased DNA mutations in coding, promoter and CTCF binding sites in RHOA mutated AITLs versus RHOA non-mutated cases as well as increased overall survival in RHOA mutated patients. In addition, single cell computational digital image analysis morphologically characterized RHOA mutated AITL cells as distinct from cells from RHOA mutation negative patients. Computational analysis of single cell morphological parameters revealed RHOA mutated cells have reduced eccentricity (more circular) compared to RHOA non-mutated AITL cells. Overall, this study expands our understanding of AITL and demonstrates that there are specific cell biologic and morphologic manifestations of RHOA mutations in cases of AITL. KeyPoints: 1) RHOA mutated AITL show distinctly increased and more deleterious mutations than RHOA unmutated cases 2) RHOA mutated T-cells have distinct morphologic features from RHOA unmutated T-cells Disclosures Ohgami: Agilent technologies: Other: received support/funding.

Detection of Multiple Recurrent Novel BCL2 Mutations Co-Occurring with BCL2 Gly101Val in Patients with Chronic Lymphocytic Leukemia on Long Term Venetoclax

The BCL2 Gly101Val mutation may be acquired in patients with chronic lymphocytic leukaemia (CLL) treated with venetoclax (VEN), leading to reduced drug binding affinity and secondary resistance. In the majority of patients, the Gly101Val mutation is subclonal within the CLL compartment consistent with the presence of alternative resistance mechanisms in CLL cells not harboring the Gly101Val mutation. To date, two Gly101Val mutated patients have been identified with co-existing candidate resistance mechanisms in Gly101Val non-mutated cells; one with BCL-XL over-expression (Blombery et al, Cancer Discov., 2019) and another with a second subclonal candidate BCL2 resistance mutation - Asp103Tyr (Tausch et al, Haematologica 2019). Given the possibility of additional resistance mechanisms, we investigated patients with progressive CLL on VEN harboring the Gly101Val mutation for the presence of additional acquired resistance mutations in BCL2. Ten patients with progressive CLL on VEN with Gly101Val mutations were identified by sensitive allele-specific droplet digital PCR. To further assess for alternative BCL2 mutations in this cohort we performed ultra-deep amplicon-based next generation sequencing (NGS) (median depth ~50,000X) targeting BCL2. An amplicon variant caller (Canary) specifically designed for low level variant calling was used (Doig et al, BMC Bioinformatics, 2017). To achieve enhanced specificity we performed digital NGS with PCR error-correction using unique molecular indexes (UMI) (QiaSEQ Targeted DNA Panel). Given the high GC content of BCL2 we also used hybridization-based NGS using a custom targeted panel (Blombery et al, BJH 2017) combined with a sensitive unpaired variant caller (GATK4/Mutect2). In 7/10 (70%) patients, BCL2 mutations in addition to the Gly101Val were detected. Recurrent mutations (detected in more than one patient) were Asp103Tyr, Asp103Glu, Arg107_Arg110dup, and Val156Asp. All additional recurrent mutations were confirmed to be absent prior to commencing VEN (sensitivity 1% variant allele frequency[VAF]). Phase-analysis of NGS reads was consistent with the presence of the additional recurrent mutations on different alleles (and therefore cells, assuming heterozygosity) to both each other and to Gly101Val. Multiple addition recurrent mutations were observed in patients in the cohort with one patient harboring three recurrent mutations in addition to the Gly101Val (Asp103Tyr, Asp103Glu, Val156Asp). In multiple patients in the cohort, the VAF of non-Gly101Val mutations exceeded that of the Gly101Val mutation. Importantly, in all patients a significant (albeit variable) proportion of CLL cells were found to be BCL2 wild-type consistent with the presence of as yet unidentified resistance mechanisms unrelated to BCL2 mutations. In one patient, two additional non-recurrent mutations were observed (Ala113Gly and Arg129Leu) in addition to Gly101Val and Val156Asp. Again, all four mutations in this patient were observed to be in mutually exclusive NGS reads. Strikingly, all of the recurrent acquired BCL2 mutated residues identified in our cohort are situated in the BCL2 binding groove that binds VEN (Figure 1). The Asp103 codon in the P4 pocket is critical for VEN binding through hydrogen bonding between its sidechain and the azaindole moiety of VEN. The Asp103Glu mutation is noteworthy given that the equivalent residue to Asp103 in BCL-XL is a Glu, which reduces VEN binding to BCL-XL. The Val156 mutation situated at the base of the P2 pocket is close to the chlorophenyl moiety of VEN and a change to Asp in this position may disrupt VEN binding. Ongoing binding experiments and modeling in cellular systems will further elucidate the mechanism and contributions of these new recurrent mutations to VEN resistance. In summary, we have extended the landscape of acquired candidate resistance mutations occurring in patients treated with VEN to include four novel recurrent BCL2 mutations. Moreover, our data are consistent with the emerging observation of multiple acquired resistance mechanisms operating in different CLL cells in a single patient contributing to an "oligoclonal" pattern of clinical relapse on VEN therapy. Figure 1 - BCL2 protein structure surface bound to venetoclax (VEN) in orange. The Asp103Tyr, Asp103Glu and Val156Asp mutation sites are shown in red and Arg107_Arg110dup region in blue Disclosures Blombery: Janssen: Honoraria; Invivoscribe: Honoraria; Novartis: Consultancy. Anderson:Walter and Eliza Hall Institute: Employment, Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.. Seymour:Acerta: Consultancy; Celgene: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Takeda: Consultancy. Huang:Genentech: Patents & Royalties: DCSH is an employee of the Walter and Eliza Hall Institute which receives milestone and royalty payments related to venetoclax. Roberts:AbbVie: Other: Unremunerated speaker for AbbVie, Research Funding; Australasian Leukaemia and Lymphoma Group: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; Walter and Eliza Hall Institute: Patents & Royalties: Institute receives royalties for venetoclax, and I receive a fraction of these.; BeiGene: Research Funding.

GENE-32. SYNTHETIC LETHAL INTERACTIONS WITH IDH1R132H IN GLIOMA STEM-LIKE CELLS

Abstract Gliomas are the most frequently diagnosed human primary brain tumors. Mutations in Isocitrate Dehydrogenase (IDH) 1 occur in the vast majority of low grade gliomas and secondary high grade glioblastomas. A single amino acid missense mutation in IDH1 at arginine 132 (R132H) is an early event in tumor development. IDH1R132H leads to the production of the oncometabolite 2-R-2-hydroxyglutarate. However the exact roles played by IDH1R132H in the development and malignant transformation of the tumors remain unclear. Further studies are required to determine optimal therapeutic strategies to target the IDH1 mutated subsets of gliomas. New generation high-throughput genetic perturbation technologies make it possible to systematically identify the genes and pathways required for the survival and proliferation of mammalian cells. Herein, we present preliminary results from a CRISPR-dCas9 derived activation to drive the transcriptional activation of more than 23,000 coding genes in both wild type and mutant IDH1 patient-derived pediatric glioma cells. Based on an average of three viability screens per cell type, we analyzed the sgRNA library representation in the IDH1 mutated and non-mutated glioma cultures after the genome wide activation. We identified 1553 candidate genes that upon gain of function trigger the death of glioma cells harboring the IDH1 mutation. The analysis of these results further pinpoints the activation of the Cyclin E1 (CCNE1), the BCL2 Antagonist/Killer 1 (BAK1) and the Homeobox B13 (HOXB13) as the most significant synthetic lethal targets in IDH1R132H glioma cells. Interestingly, results from RNAseq showed a decreased expression of these genes in IDH1 mutated compared to non-mutated glioma cells. Thus, this viability screening aims to elucidate genes that interact with IDH1R123H and play a role in tumor cell fitness. The functional analysis of these candidate genes will allow us to uncover their contribution to the progression of IDH1 mutated gliomas.

Activation of JAK/STAT Signaling in Megakaryocytes Sustains Myeloproliferation In Vivo.

The myeloproliferative neoplasms (MPN), including polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are characterized by the expansion of the erythroid, megakaryocytic, and granulocytic lineages. A common feature of these disorders is the presence of abnormal megakaryocytes, which have been implicated as causative agents in the development of bone marrow fibrosis. However, the specific contributions of megakaryocytes to MPN pathogenesis remain unclear. We used Pf4-Cre transgenic mice to drive expression of JAK2V617F in megakaryocyte lineage-committed hematopoietic cells. We also assessed the critical role of mutant megakaryocytes in MPN maintenance through cell ablation studies in JAK2V617F and MPLW515L BMT models of MPN. JAK2V617F -mutant presence in megakaryocytes was sufficient to induce enhanced erythropoiesis and promote fibrosis, which leads to a myeloproliferative state with expansion of mutant and nonmutant hematopoietic cells. The increased erythropoiesis was associated with elevated IL6 level, which was also required for aberrant erythropoiesis in vivo. Furthermore, depletion of megakaryocytes in the JAK2V617F and MPLW515L BMT models ameliorated polycythemia and leukocytosis in addition to expected effects on megakaryopoiesis. Our observations reveal that JAK/STAT pathway activation in megakaryocytes induces myeloproliferation and is necessary for MPN maintenance in vivo. These observations indicate that MPN clone can influence the behavior of the wild-type hematopoietic milieu, at least, in part, via altered production of proinflammatory cytokines and chemokines. Our findings resonate with patients who present with a clinical MPN and a low JAK2V617F allele burden, and support the development of MPN therapies aimed at targeting megakaryocytes.

Molecular analysis of glycosylphosphatidylinositol anchor deficient aerolysin resistant isolates in gulf war i veterans exposed to depleted uranium.

During the First Gulf War (1991) over 100 servicemen sustained depleted uranium (DU) exposure through wound contamination, inhalation, and shrapnel. The Department of Veterans Affairs has a surveillance program for these Veterans which has included genotoxicity assays. The frequencies of glycosylphosphatidylinositol anchor (GPIa) negative (aerolysin resistant) cells determined by cloning assays for these Veterans are reported in Albertini RJ et al. (2019: Environ Mol Mutagen). Molecular analyses of the GPIa biosynthesis class A (PIGA) gene was performed on 862 aerolysin-resistant T-lymphocyte recovered isolates. The frequencies of different types of PIGA mutations were compared between high and low DU exposure groups. Additional molecular studies were performed on mutants that produced no PIGA mRNA or with deletions of all or part of the PIGA gene to determine deletion size and breakpoint sequence. One mutant appeared to be the result of a chromothriptic event. A significant percentage (>30%) of the aerolysin resistant isolates, which varied by sample year and Veteran, had wild-type PIGA cDNA (no mutation). As described in Albertini RJ et al. (2019: Environ Mol Mutagen), TCR gene rearrangement analysis of these isolates indicated most arose from multiple T-cell progenitors (hence the inability to find a mutation). It is likely that these isolates were the result of failure of complete selection against nonmutant cells in the cloning assays. Real-time studies of GPIa resistant isolates with no PIGA mutation but with a single TCR gene rearrangement found one clone with a PIGV deletion and several others with decreased levels of GPIa pathway gene mRNAs implying mutation in other GPIa pathway genes. Environ. Mol. Mutagen. 60:470-493, 2019. © 2019 Wiley Periodicals, Inc.

Unravelling Intratumoral Heterogeneity through High-Sensitivity Single-Cell Mutational Analysis and Parallel RNA Sequencing

SummarySingle-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for resolving transcriptional heterogeneity. However, its application to studying cancerous tissues is currently hampered by the lack of coverage across key mutation hotspots in the vast majority of cells; this lack of coverage prevents the correlation of genetic and transcriptional readouts from the same single cell. To overcome this, we developed TARGET-seq, a method for the high-sensitivity detection of multiple mutations within single cells from both genomic and coding DNA, in parallel with unbiased whole-transcriptome analysis. Applying TARGET-seq to 4,559 single cells, we demonstrate how this technique uniquely resolves transcriptional and genetic tumor heterogeneity in myeloproliferative neoplasms (MPN) stem and progenitor cells, providing insights into deregulated pathways of mutant and non-mutant cells. TARGET-seq is a powerful tool for resolving the molecular signatures of genetically distinct subclones of cancer cells.

Molecular Repolarisation of Tumour-Associated Macrophages.

The tumour microenvironment (TME) is composed of extracellular matrix and non-mutated cells supporting tumour growth and development. Tumour-associated macrophages (TAMs) are among the most abundant immune cells in the TME and are responsible for the onset of a smouldering inflammation. TAMs play a pivotal role in oncogenic processes as tumour proliferation, angiogenesis and metastasis, and they provide a barrier against the cytotoxic effector function of T lymphocytes and natural killer (NK) cells. However, TAMs are highly plastic cells that can adopt either pro- or anti-inflammatory roles in response to environmental cues. Consequently, TAMs represent an attractive target to recalibrate immune responses in the TME. Initial TAM-targeted strategies, such as macrophage depletion or disruption of TAM recruitment, have shown beneficial effects in preclinical models and clinical trials. Alternatively, reprogramming TAMs towards a proinflammatory and tumouricidal phenotype has become an attractive strategy in immunotherapy. This work summarises the molecular wheelwork of macrophage biology and presents an overview of molecular strategies to repolarise TAMs in immunotherapy.

Enhanced Selective Inhibition of KRAS Mutant Hematopoietic Progenitor Cell Expansion By MEK and Bcl-2 Inhibition

Amidst the complex genetic landscape of cancer, the mutagenic and oncogenic effects caused by RAS mutations alone can be overlooked particularly in the context of hematopoietic malignancies. Therefore identifying drugs that could modulate aberrant RAS-MAPK signaling is clinically important. Here we demonstrate the utilization of an iPS cell-based system that could be adapted for compound screening to identify inhibitors with selective inhibition activity against the expansion of KRAS-mutant hematopoietic progenitor cells (HPCs).RAS-associated autoimmune lymphoproliferative disorder (RALD) is a leukemia-like disease caused by a single acquired RAS mutation. In this study, BM CD34+ cells from RALD patients were reprogrammed into iPS cells. KRAS-mutant cells could be obtained without necessitating the use of gene editing techniques. Non-mutant cells were used as isogenic controls thus permitting the assessment of unwanted toxicity against healthy cells. Constitutive activation of the RAS-MAPK pathway was confirmed by western blotting in mutant iPS cells. As expected, whole exome sequencing (WES) showed that mutant KRAS (G13C) was the only mutation present in these cells. Therefore, the inherent characteristics of mutant cell expansion can be attributed to this mutation only.Previously we reported that oncogenic KRAS enforces retention of self-renewal in human iPS cells (Kubara et al. Stem Cell Reports. 2018) due to constitutive activation of the KRAS-MAPK signaling pathway. Notably mutant iPS cells retained pluripotency marker expression even in the absence of bFGF, but not isogenic control cells. When we tested compounds with different mechanisms of action, direct MEK inhibition was the most effective in triggering a loss of pluripotency marker expression in mutant iPS cells in the absence of bFGF. This method permits rapid determination of compounds that could modulate aberrant KRAS-MAPK signaling. However, further verification is required to determine whether inhibitory effects on mutant HPC expansion could also be observed.Next, differentiated multi-potent (Lin-34+43+) HPCs from iPS cells were expanded in “standard” ex vivo culture conditions using SCF, TPO Flt3L and IL-3. This favored the expansion of control cells. We also found “selective” culture conditions that could selectively expand mutant HPCs. RNA-seq analysis on pre-expanded and expanded HPCs revealed the unique molecular signature of expanding mutant cells, meaning genes that showed upregulation in both “standard” and “selective” culture conditions. There was upregulation of CCND1 and CDK4 in mutant HPCs, which facilitates progression to the S phase of the cell cycle. This was also accompanied by upregulation of p16INK4a and p21Cip1, which is characteristic of senescent cells. Indeed, it could be shown that expanded mutant HPCs acquired senescence features such as increased p16 INK4a and Bcl-xL expression at the protein level and increased SA-b-gal activity compared with control cells. Gene correction of KRAS (G13C) using CRISPR/Cas9 reversed these phenotypes. Therefore CCND1/CDK4 and Bcl-2 may be potential inhibitory targets.We developed a high-throughput screening method whereby differentiated HPCs were sorted directly into 96-well plates and following expansion, cell numbers were quantified by luminescence detection of ATP. First we screened drugs for selective activity (IC50 value for mutant cells lower than control cells). Consistent with the iPS cell pluripotency assay, we identified Trametinib (MEK), as having selective effects against mutant HPCs in addition to Palbociclib (CCND1/CDK4) and Navitoclax (Bcl-2). When used in combination, the selective inhibitory effect was enhanced without compromising the viability of control cells.To test these drugs in an in vivo setting, highly purified transduced (KRAS or control) mouse HSCs (Lin-CD34-KSL) were transplanted in a competitive repopulation assay. Recipient mice were given Trametinib and Navitoclax. In the KRAS group, donor cells contributed to a lower level of chimerism. We also developed the use of droplet digital PCR (ddPCR) to monitor the percentage of mutant cells within bulk BM CD34+ cells (mutant and isogenic control) when cultured in the presence of inhibitors. Taken together, our study offers novel insights into mutant KRAS-driven HPC expansion and demonstrate an efficient platform for drug screening. DisclosuresKubara:Eisai Co., Ltd.: Employment. Yamazaki:Eisai Co., Ltd.: Employment.

Dietary HNE and other lipid oxidation products in the development of colorectal carcinogenesis

Based on epidemiological studies and meta-analyses, the World Cancer Research Fund (WCRF) panel stated that red meat is a convincing cause of colorectal cancer. Recently, the IARC/WHO classified red meat consumption as “probably carcinogenic to humans” (Group 2A). Red meat contains high concentrations of heme iron that induces lipid peroxidation in the colon lumen and the subsequent formation of secondary lipid oxidation products such as 4-hydroxynonenal (HNE). HNE was found in foodstuffs in relatively high concentration, particularly in meat and processed meat. Moreover, HNE formation possibly occurs in the digestive tract when rats receive heme iron and a PUFA-rich oil at the same time. In vivo studies in rats show that the development of preneoplastic lesions is well correlated with heme-iron and HNE content of the diet, and also with the urinary excretion of the major metabolite of HNE, the mercapturic acid of 1,4-dihydroxynonene (DHN-MA). Secondary oxidation products of PUFA, and especially HNE, could be one of the missing links between heme iron, enhanced luminal lipid peroxidation and colon cancer development. In vitro studies in mouse epithelial colon immortalized cells show that the cells mutated on the Apc (Adenomatous polyposis coli) gene, an early and frequent mutation during the development of human colorectal cancer, were more resistant to HNE than non-mutated cells. This resistance is due to a better metabolization capacity of those cells towards HNE. The enzymes involved are under the transcriptional control of Nrf2/antioxidant responsive element (ARE) pathway, a pathway that is upregulated in Apc mutated cells. Nrf2 natural inducers, as curcumin, have a protective effect both in vitro and in vivo.

Clonal heterogeneity of acute myeloid leukemia treated with the IDH2 inhibitor enasidenib.

Mutations in the gene encoding isocitrate dehydrogenase 2 (IDH2) occur in several types of cancer, including acute myeloid leukemia (AML). In model systems, mutant IDH2 causes hematopoietic differentiation arrest. Enasidenib, a selective small-molecule inhibitor of mutant IDH2, produces a clinical response in 40% of treated patients with relapsed/refractory AML by promoting leukemic cell differentiation. Here, we studied the clonal basis of response and acquired resistance to enasidenib treatment. Using sequential patient samples, we determined the clonal structure of hematopoietic cell populations at different stages of differentiation. Before therapy, IDH2-mutant clones showed variable differentiation arrest. Enasidenib treatment promoted hematopoietic differentiation from either terminal or ancestral mutant clones; less frequently, treatment promoted differentiation of nonmutant cells. Analysis of paired diagnosis/relapse samples did not identify second-site mutations in IDH2 at relapse. Instead, relapse arose by clonal evolution or selection of terminal or ancestral clones, thus highlighting multiple bypass pathways that could potentially be targeted to restore differentiation arrest. These results show how mapping of clonal structure in cell populations at different stages of differentiation can reveal the response and evolution of clones during treatment response and relapse.

Abstract 1429: MYH8 R1292X: A novel mutation in relapsed AML induces EMT features and poor prognosis

Abstract Introduction: Recent advances in tumor genomic analysis have led to the discovery of NPM1, FLT3, CEBPA, MLL, NRAS, and RUNX1 alterations as the cause of AML. Nevertheless, there is a limit to the treatment and clarification of AML, and research for the identification of novel genetic alterations that cause AML is actively underway. Materials and methods: In this study, we performed Whole exome sequencing (WES) with 53 AML patient's samples and conducted targeted re-sequencing using 391 AML patient's samples based on locus of somatic mutation that were found by WES. For functional validation of novel oncogenic mutations, we used CRISPR-Cas9 system to generate knock-in (KI) cell line. For characterization of mutant cells, we performed proliferation assay, cell cycle assay, adhesion assay, and wound healing assay. Epithelial to mesenchymal transition (EMT) markers were checked by western blotting. Results: Using WES and targeted resequencing, we could identify MYH8 R1292X novel mutation as recurrent potentially oncogenic mutation. Additional validation using separate AML cohort revealed MYH8 R1292X variants in four AML patients, suggesting that MYH8 R1292X is potential oncogenic mutation. In functional validation using KI cell line, we could not find change in morphology of KI cells. However, there was a difference in proliferation – the rate of proliferation was faster in KI cells than in cells without mutation. In the cell cycle assay, the mutant cells showed more S phase DNA than the non-mutant cells. Wound healing assay showed that the mutant cells had higher migration ability and lowered the ability of adhesion in comparison. PCR and western blot showed that EMT markers except vimentin increased in mutant cells.Survival analysis based on TCGA data showed that both the overall survival and the disease-free survival curves were significantly different according to MYH8 alterations. Conclusion: Taken together, we conclude that the novel alteration MYH8 R1292X is associated with recurrent AML and poor prognosis by increasing migration, and inducing an increase in EMT markers. Citation Format: Hyejoo Park, Daeyoon Kim, Dongchan Kim, Yungyeong Park, Youngil Koh, Sung-Soo Yoon. MYH8 R1292X: A novel mutation in relapsed AML induces EMT features and poor prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1429.

PO-103 Regulation of prostate cancer cell growth and signalling by exercise

IntroductionMalignant mesothelioma (MM) is an aggressive cancer of the mesothelium. MM has a poor prognosis with a median post-diagnosis survival of 12 months. Currently, MM is treated with platinum-based chemotherapy, resulting in side-effects to the patient. Hence, efforts have been focused on genetically analysing MM tumours to improve treatment specificity. Genetically, MMs are characterised by the loss of key tumour suppressor genes such as NF2. NF2 functions as a member of the Hippo signalling pathway. The Hippo pathway consists of a tumour-suppressive kinase cascade which inactivates the proto-oncogenic transcription factors YAP and TAZ. NF2 up-regulates the Hippo pathway kinases to reduce the activity of YAP/TAZ. Hence, NF2 mutant MMs may exhibit hyperactivity of YAP/TAZ, driving oncogenic processes. So, YAP/TAZ may be ideal therapeutic targets for treating NF2 mutant MMs.Material and methodsA panel of 7 MM cell lines and one wildtype mesothelial cell line were used to assess the potential for selectively targeting YAP/TAZ to treat MMs with mutations in NF2 or in the Hippo pathway kinases LATS1/2. YAP and TAZ activity was abrogated in the cell lines using siRNA-mediated knockdown or using chemical inhibition with the drug compounds Verteporfin or Simvastatin. The viability of cell lines 96 hours post YAP/TAZ inhibition was measured using an Alamar blue viability assay. Statistical comparisons of cellular responses to YAP/TAZ inhibition were conducted using an unpaired student’s t-test.Results and discussionsThe effect of YAP/TAZ inhibition on the viability of a panel of 7 MM cell lines and a wildtype mesothelial cell line (Met5A) was assessed. Hippo pathway mutant cell lines showed the highest sensitivity to YAP/TAZ knockdown, however, two non-mutant MM cells also showed considerable sensitivity, indicating that there wasn’t a statistically significant dependence of mutant cell lines on YAP/TAZ for viability. Mutant cell lines did not show selective sensitivity to Simvastatin or Verteporfin, suggesting that these compounds may not be ideal treatment options. However, these observations were likely confounded by the heterogeneity of the panel of cell lines. As such, an isogenic system of NF2 mutant Met5A cells was developed to validate the findings.ConclusionBased on our findings, YAP and TAZ may not be ideal targets for treating Hippo pathway mutant MMs. However, the use of an NF2 mutant isogenic MM cell line system will provide a definitive outlook on their feasibility as therapeutic candidates.

PO-100 Targeting YAP and TAZ to treat hippo pathway mutant malignant mesotheliomas

IntroductionMalignant mesothelioma (MM) is an aggressive cancer of the mesothelium. MM has a poor prognosis with a median post-diagnosis survival of 12 months. Currently, MM is treated with platinum-based chemotherapy, resulting in side-effects to the patient. Hence, efforts have been focused on genetically analysing MM tumours to improve treatment specificity. Genetically, MMs are characterised by the loss of key tumour suppressor genes such as NF2. NF2 functions as a member of the Hippo signalling pathway. The Hippo pathway consists of a tumour-suppressive kinase cascade which inactivates the proto-oncogenic transcription factors YAP and TAZ. NF2 up-regulates the Hippo pathway kinases to reduce the activity of YAP/TAZ. Hence, NF2 mutant MMs may exhibit hyperactivity of YAP/TAZ, driving oncogenic processes. So, YAP/TAZ may be ideal therapeutic targets for treating NF2 mutant MMs.Material and methodsA panel of 7 MM cell lines and one wildtype mesothelial cell line were used to assess the potential for selectively targeting YAP/TAZ to treat MMs with mutations in NF2 or in the Hippo pathway kinases LATS1/2. YAP and TAZ activity was abrogated in the cell lines using siRNA-mediated knockdown or using chemical inhibition with the drug compounds Verteporfin or Simvastatin. The viability of cell lines 96 hours post YAP/TAZ inhibition was measured using an Alamar blue viability assay. Statistical comparisons of cellular responses to YAP/TAZ inhibition were conducted using an unpaired student’s t-test.Results and discussionsThe effect of YAP/TAZ inhibition on the viability of a panel of 7 MM cell lines and a wildtype mesothelial cell line (Met5A) was assessed. Hippo pathway mutant cell lines showed the highest sensitivity to YAP/TAZ knockdown, however, two non-mutant MM cells also showed considerable sensitivity, indicating that there wasn’t a statistically significant dependence of mutant cell lines on YAP/TAZ for viability. Mutant cell lines did not show selective sensitivity to Simvastatin or Verteporfin, suggesting that these compounds may not be ideal treatment options. However, these observations were likely confounded by the heterogeneity of the panel of cell lines. As such, an isogenic system of NF2 mutant Met5A cells was developed to validate the findings.ConclusionBased on our findings, YAP and TAZ may not be ideal targets for treating Hippo pathway mutant MMs. However, the use of an NF2 mutant isogenic MM cell line system will provide a definitive outlook on their feasibility as therapeutic candidates.

Selection for increased quorum-sensing cooperation in Pseudomonas aeruginosa through the shut-down of a drug resistance pump

The opportunistic pathogen Pseudomonas aeruginosa employs a hierarchical quorum-sensing network to regulate virulence factor production that cooperatively benefit the population at a cost to the individual. It has been argued that the evolution of a cooperative mutant in a quorum sensing-suppressed population would be hampered through its exploitation by neighboring non-mutant cells. It remains unclear whether mechanisms which overcome this exploitation exist. Here we investigate the regain of quorum-sensing cooperation by evolving a mutant of the lasR master quorum-sensing regulator. The mutant regained partial cooperative growth through null mutations in mexT, which codes for an activator of the MexEF-OprN multidrug-resistant pump. We find that these mutations enhance cooperative growth in both the lasR mutant and wild-type backgrounds through the activation of the RhlIR system. We show that the regain of cooperation in mexT mutants is mediated by the reduction in MexEF-OprN activity, whereas an additional source of private benefit is mostly mexEF-oprN-independent. Finally, we show that addition of antibiotics for which resistance is mediated by MexEF-OprN prevents the selection of increased cooperation at sub-MIC concentrations. MexT, therefore, not only links private and public goods, but also exposes conflicts between selection for antibiotic resistance and enhanced cooperation.

CBL mutation and MEFV single-nucleotide variant are important genetic predictors of tumor reduction in glucocorticoid-treated patients with chronic myelomonocytic leukemia.

Glucocorticoid (GC) therapy occasionally relieves tumor-related fever and promotes tumor reduction in patients with chronic myelomonocytic leukemia (CMML). A mutation analysis of 24 patients with CMML revealed the relationship of GC effectiveness, defined as a monocyte reduction of > 50% within 3days of methylprednisolone administration, with the MEFV single-nucleotide variant (SNV) and CBL mutation. Lipopolysaccharide-stimulated monocytes harboring MEFV E148Q produced greater amounts of IL-1β and TNF-α than did wild-type monocytes; this was effectively suppressed by GC. Primary CMML cells harboring the MEFV SNV and CBL mutation, and the myelomonocytic leukemia cell line GDM-1, harboring the CBL mutation, were both more significantly suppressed than non-mutated cells following GC treatment in the presence of GM-CSF. A loss-of-function CBL mutation prolonged STAT5 phosphorylation after GM-CSF stimulation, which was rapidly terminated in both patient samples and GDM-1 cells. In conclusion, GC therapy effectively treats CMML cells harboring the MEFV SNV and CBL mutation by reducing inflammatory cytokine production and terminating prolonged STAT5 phosphorylation in the GM-CSF signaling pathway.

Synthetic Lethality Triggered by Combining Olaparib with BRCA2-Rad51 Disruptors.

In BRCA2-defective cells, poly(adenosine diphosphate [ADP]-ribose) polymerase inhibitors can trigger synthetic lethality, as two independent DNA-repairing mechanisms are simultaneously impaired. Here, we have pharmacologically induced synthetic lethality, which was triggered by combining two different small organic molecules. When administered with a BRCA2-Rad51 disruptor in nonmutant cells, Olaparib showed anticancer activity comparable to that shown when administered alone in BRCA2-defective cells. This strategy could represent an innovative approach to anticancer drug discovery and could be extended to other synthetic lethality pathways.

H-Ferritin-nanocaged olaparib: a promising choice for both BRCA-mutated and sporadic triple negative breast cancer

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

NRAS mutations in cutaneous T cell lymphoma (CTCL) sensitize tumors towards treatment with the multikinase inhibitor Sorafenib.

Therapy of cutaneous T cell lymphoma (CTCL) is complicated by a distinct resistance of the malignant T cells towards apoptosis that can be caused by NRAS mutations in late-stage patients. These mutations correlate with decreased overall survival, but sensitize the respective CTCL cells towards MEK-inhibition-induced apoptosis which represents a promising novel therapeutic target in CTCL. Here, we show that the multi-kinase inhibitor Sorafenib induces apoptosis in NRAS-mutated CTCL cells. CTCL cell lines and to a minor extent primary T cells from Sézary patients without NRAS mutations are also affected by Sorafenib-induced apoptosis suggesting a sensitizing role of NRAS mutations for Sorafenib-induced apoptosis. When combining Sorafenib with the established CTCL medication Vorinostat we detected an increase in cell death sensitivity in CTCL cells. The combination treatment acted synergistically in apoptosis induction in both non-mutant and mutant CTCL cells. Mechanistically, this synergistic apoptosis induction by Sorafenib and Vorinostat is based on the downregulation of the anti-apoptotic protein Mcl-1, but not of other Bcl-2 family members. Taken together, these findings suggest that Sorafenib in combination with Vorinostat represents a novel therapeutic approach for the treatment of CTCL patients.

Combination Targeted Therapy to Disrupt Aberrant Oncogenic Signaling and Reverse Epigenetic Dysfunction in IDH2- and TET2-Mutant Acute Myeloid Leukemia.

Genomic studies in acute myeloid leukemias (AML) have identified mutations that drive altered DNA methylation, including TET2 and IDH2 Here, we show that models of AML resulting from TET2 or IDH2 mutations combined with FLT3ITD mutations are sensitive to 5-azacytidine or to the IDH2 inhibitor AG-221, respectively. 5-azacytidine and AG-221 treatment induced an attenuation of aberrant DNA methylation and transcriptional output and resulted in a reduction in leukemic blasts consistent with antileukemic activity. These therapeutic benefits were associated with restoration of leukemic cell differentiation, and the normalization of hematopoiesis was derived from mutant cells. By contrast, combining AG-221 or 5-azacytidine with FLT3 inhibition resulted in a reduction in mutant allele burden, progressive recovery of normal hematopoiesis from non-mutant stem-progenitor cells, and reversal of dysregulated DNA methylation and transcriptional output. Together, our studies suggest combined targeting of signaling and epigenetic pathways can increase therapeutic response in AML.Significance: AMLs with mutations in TET2 or IDH2 are sensitive to epigenetic therapy through inhibition of DNA methyltransferase activity by 5-azacytidine or inhibition of mutant IDH2 through AG-221. These inhibitors induce a differentiation response and can be used to inform mechanism-based combination therapy. Cancer Discov; 7(5); 494-505. ©2017 AACR.See related commentary by Thomas and Majeti, p. 459See related article by Yen et al., p. 478This article is highlighted in the In This Issue feature, p. 443.