Non-mutated Cells Research Articles

Roles of Notch Signaling in the Tumor Microenvironment.

The Notch signaling pathway is an architecturally simple signaling mechanism, well known for its role in cell fate regulation during organ development and in tissue homeostasis. In keeping with its importance for normal development, dysregulation of Notch signaling is increasingly associated with different types of tumors, and proteins in the Notch signaling pathway can act as oncogenes or tumor suppressors, depending on the cellular context and tumor type. In addition to a role as a driver of tumor initiation and progression in the tumor cells carrying oncogenic mutations, it is an emerging realization that Notch signaling also plays a role in non-mutated cells in the tumor microenvironment. In this review, we discuss how aberrant Notch signaling can affect three types of cells in the tumor stroma—cancer-associated fibroblasts, immune cells and vascular cells—and how this influences their interactions with the tumor cells. Insights into the roles of Notch in cells of the tumor environment and the impact on tumor-stroma interactions will lead to a deeper understanding of Notch signaling in cancer and inspire new strategies for Notch-based tumor therapy.

Postmortem Diagnosis of the Proteus Syndrome by Next Generation Sequencing of Affected Brain Tissue.

We report a case of a somatic overgrowth syndrome diagnosed at forensic autopsy with the aid of next generation sequencing as Proteus syndrome. Somatic overgrowth syndromes result from spontaneous somatic mutations that arise early in development and display a mosaic pattern of expression in patient tissues. Due to the temporal and anatomic heterogeneity of these syndromes, phenotypes vary widely, resulting in clinical overlap. Furthermore, the variable ratio of mutated to nonmutated cells in patient tissue can result in low-level mutations that could be missed using Sanger sequencing. Due to these factors, recent literature points to next generation sequencing (NGS) as an adjunct to diagnosis of these rare entities. A male in his fourth decade of life presented to our forensic autopsy service with physical features suggestive of a somatic overgrowth syndrome. Due to the paucity of clinical information accompanying the individual, a definitive diagnosis based on physical characteristics, alone, was not possible. Next generation sequencing of affected formalin-fixed and paraffin-embedded brain tissue confirmed the presence of the variant in AKT1 (c.49G>A, p.Glu17Lys, in 14.13% of reads) found in Proteus syndrome. To our knowledge, this is the first report of the mosaic variant of AKT1 detected in brain tissue and the first reported case of a postmortem diagnosis of Proteus syndrome with the aid of NGS. We conclude that NGS can be used as an adjunctive method to support a specific diagnosis among the somatic overgrowth syndromes postmortem in the absence of sufficient clinical history.

Analysis of Pathogenic Pseudoexons Reveals Novel Mechanisms Driving Cryptic Splicing.

Understanding pre-mRNA splicing is crucial to accurately diagnosing and treating genetic diseases. However, mutations that alter splicing can exert highly diverse effects. Of all the known types of splicing mutations, perhaps the rarest and most difficult to predict are those that activate pseudoexons, sometimes also called cryptic exons. Unlike other splicing mutations that either destroy or redirect existing splice events, pseudoexon mutations appear to create entirely new exons within introns. Since exon definition in vertebrates requires coordinated arrangements of numerous RNA motifs, one might expect that pseudoexons would only arise when rearrangements of intronic DNA create novel exons by chance. Surprisingly, although such mutations do occur, a far more common cause of pseudoexons is deep-intronic single nucleotide variants, raising the question of why these latent exon-like tracts near the mutation sites have not already been purged from the genome by the evolutionary advantage of more efficient splicing. Possible answers may lie in deep intronic splicing processes such as recursive splicing or poison exon splicing. Because these processes utilize intronic motifs that benignly engage with the spliceosome, the regions involved may be more susceptible to exonization than other intronic regions would be. We speculated that a comprehensive study of reported pseudoexons might detect alignments with known deep intronic splice sites and could also permit the characterisation of novel pseudoexon categories. In this report, we present and analyse a catalogue of over 400 published pseudoexon splice events. In addition to confirming prior observations of the most common pseudoexon mutation types, the size of this catalogue also enabled us to suggest new categories for some of the rarer types of pseudoexon mutation. By comparing our catalogue against published datasets of non-canonical splice events, we also found that 15.7% of pseudoexons exhibit some splicing activity at one or both of their splice sites in non-mutant cells. Importantly, this included seven examples of experimentally confirmed recursive splice sites, confirming for the first time a long-suspected link between these two splicing phenomena. These findings have the potential to improve the fidelity of genetic diagnostics and reveal new targets for splice-modulating therapies.

Glucosylceramide Synthase Is a Novel Biomarker of Midostaurin-Induced Cytotoxicity in Non-Mutant FLT3 Positive Acute Myeloid Leukemia Cells

Objective: Glucosylceramide (GC) synthesized by glucosylceramide synthase (GCS) favors cell survival and proliferation in many cancers. However, it’s role in Fms-like tyrosine kinase 3 (FLT3) non-mutant Acute Myeloid Leukemia (AML) pathogenesis is not clarified. Midostaurin, a multi-kinase inhibitor, clinically benefits FLT3-mutated AML, however, its clinical efficacy is under-estimated in FLT3 non-mutant AML. This study aimed to investigate the efficacy of combination of midostaurin with GCS inhibitor in FLT3 AML cell carrying wild-type FLT3 and the underlying molecular mechanisms. Material and Method: Cytotoxic and cytostatic effects of midostaurin, PDMP (GCS inhibitor) alone and in combination on THP1 cells were determined by MTT assay and flow cytometric propidium iodide (PI) staining, respectively. Calcusyn software was used to calculate combination indexes (CIs). GCS expression was checked by western blot. Results: Midostaurin downregulated GCS. Simultaneous inhibition of FLT3 and GCS resulted in suppression of cell proliferation as compared to untreated control. Combinations showed synergistic cytotoxic effects (CI<1). Co-treatments increased cell cycle population at G2/M phase. Conclusion: Inhibition of GCS enhances the efficacy of midostaurin in FLT3 non-mutant AML, which could be a novel therapeutic approach to increase midostaurin’s limited usage in the clinic after detailed mechanistic studies.

Targeting Control of Cell Cycle Enhances the Activity of Conventional Chemotherapy in Chemotherapy-Resistant Acute Myeloid Leukemia

Chemotherapy-resistant acute myeloid leukemia (AML) manifesting as primary refractory or relapsed disease carries a dismal prognosis and is often driven by clonal evolution. We have performed a genome-wide CRISPR knockout screen investigating resistance to conventional AML therapy, cytarabine and doxorubicin (AraC/Dox), in 2 independent human AML cell lines. Chemoresistant populations were enriched with gRNAs disrupting cell cycle arrest genes, including cyclin dependent kinase inhibitor 2A (CDKN2A), checkpoint kinase 2 (CHEK2) and TP53. Here, we validate the direct contribution of these genes to chemoresistance and demonstrate that rationally designed therapeutic regimens targeting cell cycle enhances chemotherapy response in AML.Using CRISPR-mediated gene editing, we individually inactivated CDKN2A and CHEK2 in Cas9-expressing OCI-AML3 and MV4-11 cells. CDKN2A- and CHEK2-deficient cells demonstrated proliferative advantage in the presence of AraC/Dox in co-culture competition assays, confirming direct contribution of these gene knockouts to chemoresistance. Nil to modest reductions in apoptosis were seen in CDKN2A- and CHEK2-deficient cells treated with AraC/Dox for 72 hours compared to unedited controls. However, failure of accumulation of cells in the non-cycling G 0/G 1 proportion was seen in these edited cells after chemotherapy, corresponding with maintained DNA synthesis, as measured by BrdU incorporation, and a failure to downregulate phospho-Rb protein expression, indicating ongoing active cell cycling in spite of chemotherapy. These results confirm failure of cell cycle arrest as the major mechanism of resistance with inactivation of CDKN2A or CHEK2.To assess relevance of these genes in chemotherapy response in human AML, we analysed the effect of CDKN2A expression on prognosis. Reduced expression of CDKN2A conferred inferior overall survival in 3 independent clinical cohorts. Additionally, downregulation of CDKN2A and an increase in downstream cell cycling effector, cyclin-dependent kinase 6 (CDK6) was seen at relapse in paired diagnosis-relapse AML samples (Li et al. 2016, Nature Medicine). Further, CHEK2 mutations in clonal hematopoiesis are enriched in patients with solid organ cancers following chemo- or radiotherapy, functionally demonstrating chemoresistance over non-mutated cells. These data suggest CDKN2A and CHEK2 loss-of-function is relevant in promoting chemoresistance in human hematopoiesis.We therefore investigated whether therapeutically targeting cell cycle control pathways that converge on the G 1/S restriction point could synergise with cytotoxic chemotherapy, potentially circumventing chemoresistance. The addition of MDM2 inhibitor, nutlin-3a with AraC/Dox achieved striking synergism in TP53-competent cell lines in reducing viability and promoting apoptosis. Inhibition of CDK4/6 with palbociclib alone induced cell cycle arrest without apoptosis, however combination therapy with AraC/Dox significantly potentiated apoptosis. To functionally validate the role of CDKN2A itself, we examined the inhibitor of histone acetyltransferase KAT6A, WM-1119 (Baell et al. 2018, Nature), which transcriptionally upregulates CDKN2A. WM-1119 had anti-leukemic activity across multiple cell lines, corresponding with down regulation of cell cycling and upregulation of senescence signatures. The anti-leukemic activity was prevented by CDKN2A inactivation, indicating that CDKN2A upregulation is essential for WM-1119's anti-leukemic effect. WM-1119 enhanced activity of AraC/Dox in multiple cell contexts, mediated by cooperative induction of CDKN2A. Its efficacy and synergy with AraC/Dox was further demonstrated in MLL-AF9 primary murine AML ex vivo, demonstrating broader applicability of the approach.Our findings reveal defects in cell cycle arrest pathways as clinically relevant contributors to chemoresistance in AML. Combining agents to target cell cycle components improved in vitro anti-leukemic activity of chemotherapy and warrants further exploration of their translational potential. DisclosuresBullinger: Amgen: Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Menarini: Consultancy; Hexal: Consultancy; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Consultancy, Honoraria; Astellas: Honoraria; Gilead: Consultancy; Daiichi Sankyo: Consultancy, Honoraria; Seattle Genetics: Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Sanofi: Honoraria; Abbvie: Consultancy, Honoraria. Lane: BMS: Consultancy, Research Funding; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria; Novartis: Consultancy; Geron: Consultancy.

U2af1 is a haplo-essential gene required for hematopoietic cancer cell survival in mice.

Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained WT allele is expressed, suggesting that mutant hematopoietic cells may require the residual WT allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knockout mice were similar to those in control mice, but that deletion of the WT allele in U2AF1(S34F) heterozygous mutant-expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of WT U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1(S34F)-expressing cells were also more sensitive to reduced expression of WT U2AF1 than nonmutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the WT U2af1 allele was deleted compared with when it was not deleted. These results suggest that selectively targeting the WT U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.

Tumorigenesis and cell competition in Drosophila in the absence of polyhomeotic function

Cell competition is a homeostatic process that eliminates by apoptosis unfit or undesirable cells from animal tissues, including tumor cells that appear during the life of the organism. In Drosophila there is evidence that many types of oncogenic cells are eliminated by cell competition. One exception is cells mutant for polyhomeotic (ph), a member of the Polycomb family of genes; most of the isolated mutant ph clones survive and develop tumorous overgrowths in imaginal discs. To characterize the tumorigenic effect of the lack of ph, we first studied the growth of different regions of the wing disc deficient in ph activity and found that the effect is restricted to the proximal appendage. Moreover, we found that ph-deficient tissue is partially refractory to apoptosis. Second, we analyzed the behavior of clones lacking ph function and found that many suffer cell competition but are not completely eliminated. Unexpectedly, we found that nonmutant cells also undergo cell competition when surrounded by ph-deficient cells, indicating that within the same tissue cell competition may operate in opposite directions. We suggest two reasons for the incompleteness of cell competition in ph mutant cells: 1) These cells are partially refractory to apoptosis, and 2) the loss of ph function alters the identity of imaginal cells and subsequently their cell affinities. It compromises the winner/loser interaction, a prerequisite for cell competition.

Oncogene-induced senescence in hematopoietic progenitors features myeloid restricted hematopoiesis, chronic inflammation and histiocytosis

ABSTRACTActivating mutations in the BRAF-MAPK pathway have been reported in histiocytoses, hematological inflammatory neoplasms characterized by multi-organ dissemination of pro-inflammatory myeloid cells. Here, we generate a humanized mouse model of transplantation of human hematopoietic stem and progenitor cells (HSPCs) expressing the activated form of BRAF (BRAFV600E). All mice transplanted with BRAFV600E-expressing HSPCs succumb to bone marrow failure, displaying myeloid-restricted hematopoiesis and multi-organ dissemination of aberrant mononuclear phagocytes. At the basis of this aggressive phenotype, we uncover the engagement of a senescence program, characterized by DNA damage response activation and a senescence-associated secretory phenotype, which affects also non-mutated bystander cells. Mechanistically, we identify TNFα as a key determinant of paracrine senescence and myeloid-restricted hematopoiesis and show that its inhibition dampens inflammation, delays disease onset and rescues hematopoietic defects in bystander cells. Our work establishes that senescence in the human hematopoietic system links oncogene-activation to the systemic inflammation observed in histiocytic neoplasms.

Interactions of BRCA1-mutated Breast Cancer Cell Lines with Adipose-derived Stromal Cells (ADSCs)

Lipofilling may constitute a technique to assist reconstruction of breasts following prophylactic mastectomy for patients with mutated BRCA1 or BRCA2 genes. However, to date it is not clear whether adipose-derived stromal cells (ADSCs) increase the risk of tumor initiation and progression in this situation. Therefore, the interactions of BRCA1 mutated breast cancer cell lines with normal ADSCs were investigated in the present study. Characteristics of MDA-MB-436 (BRCA1 c.5277 + 1G > A) and HCC1937 (BRCA1 p.Gln1756.Profs*74) were compared to MDA-MB-231 and T47D BRCA1/2 wild-type breast cancer cell lines. ADSCs were cultivated from lipoaspirates of a panel of BRCA1/2- wildtype patients. Interactions of conditioned medium (CM) of these cells with the breast cancer lines were studied using proliferation and migration assays as well as adipokine expression western blot arrays. CM of ADSCs exhibit a dose-dependent stimulation of the proliferation of the breast cancer cell lines. However, of the ADSC preparations tested, only 1 out of 18 samples showed a significant higher stimulation of BRCA1-mutated MDA-MB-436 versus wildtype MDA-MB-231 cells, and all CM revealed lower stimulatory activity for BRCA1-mutated HCC1937 versus wildtype T47D cells. Additionally, migration of breast cancer cells in response to CM of ADSCs proved to be equivalent or slower for BRCA1/2 mutated versus nonmutated cancer cells and, with exception of angiopoietin-like 2, induced expression of adipokines showed no major difference. Effects of media conditioned by normal ADSCs showed largely comparable effects on BRCA1-mutated and wildtype breast cancer cell lines thus advocating lipofilling, preferentially employing allogeneic non-mutated ADSCs.

Imbalance between Tolerant and Cytotoxic Functional Subsets of NK Cells in a JAK2V617F Myeloproliferative Neoplasm Model

Natural killer (NK) cells are lymphocytes that play a critical role in both innate and adaptive immune responses. It has been shown that CD27 and CD11b are antigens expressed in the last stage of NK cells development and allow the classification of NK cells into three functional subsets. Tolerant NK cells are double negative (DN) for CD27 and CD11b, have an immature phenotype and present low secretory and cytotoxic potential. The secretory NK cells are CD27 positive, have a great ability to release cytokines, but do not exhibit cytotoxicity properties. The cytotoxic NK cells express CD11b, release granzymes and perforins, but are less capable of secreting cytokines. While cytotoxic NK cells are essential for the natural defenses against infected or malignant cells, secretory NK cells, when activated, produce different cytokines, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10 and others, which may modify the functions or other hematopoietic cells including the hematopoietic stem cells (HSC). We hypothesized that deregulation of the balance between secretory or cytotoxic subtypes of NK cells may favor HSC malignant transformation and contribute to stem cell-derived diseases. In this regard, few studies have shown that NK cells are defective in chronic myeloid leukemia (CML). In contrast to CML, the role of NK cells in BCR-ABL1 negative-myeloproliferative neoplasms (MPN) is currently unexplored. We aimed to study the activity of NK cells and their subsets in murine primary cells from MPN. NK cells subsets were quantified by flow cytometry by the use of spleen cells from a pre-established conditional vavCre knockin Jak2V617F (Jak2VF) murine model. Besides genotyping to identify controls (Jak2 wt/wt vavCre+, or Jak2WT) and Jak2-mutated mice (Jak2 wt/V617F vavCre+, Jak2VF), the presence of polycythemia and splenomegaly was confirmed in the animals with MPN. Mice (n=5 per group) were euthanized between 8-12 weeks of age. For immunophenotyping, spleen cells were isolated, submitted to red cell lyse, and stained with fluorescence-conjugated antibodies against Ter119, CD19, CD4, CD8, CD3, NK1.1 and CD27 and CD11b. All the stainings were performed at 4°C for 20 minutes and acquisition was performed in a FACSCanto™II flow cytometer (BD Biosciences). Data was analyzed by the use of the Flowjo software version X. When compared to Jak2WT controls, the Jak2VF mice had lower numbers of NK cells (1,447 x 106 versus 0,381 x 106 p<0.05) and T cells (0,638 x 106 versus 0,158 x 106 of the spleen; p<0.05) among the total splenocytes. The analysis of NK subsets showed an increase in the percentages of DN NK cells (CD27-CD11b-) in the spleen of Jak2VF mice as compared to controls (14.84% versus 4.97%; p<0.05). CD27+ immature and mature secretory NK (CD27+CD11b-/+) cells also seemed to be increased in Jak2-mutated mice but this data did not reach statistical significance (37.43% versus 17.65%; p=0.1). On the other hand, when compared to Jak2WT controls, the Jak2VF mice presented lower percentages of CD11b+ cytotoxic NK cells (77.07% versus 47.80% of the spleen cells; p<0.05). In agreement, when the expression of NK receptors was analyzed by flow cytometry, the inhibitory NKG2A receptor was found to be higher expressed in Jak2VF NK cells as compared to non-mutated cells, while the expression of the activatory receptors Ly49H and Ly49D was reduced in Jak2-mutated NK cells. Of note, low levels of the Jak2V617F mutation were detected in sorted NK cells isolated from Jak2VF mice as compared to controls by Real Time quantitative RT-PCR, thus suggesting that the distribution between NK cell subtypes observed here is associated with the JAK2 mutational status. In summary, when compared to controls, Jak2-mutated mice presented an increased proportion of tolerant NK cells, previously described as immature cells with low secretory and cytotolytic potential. To date, CD11b-/CD27- tolerant cells have been observed among solid tumor-infiltrating NK cells, but have not been previously associated with MPN. Moreover, an inverted ratio between the secretory and cytotoxic NK subsets was also detected in Jak2VF animals, suggesting that a cytotoxic deficiency occurs and may contribute to the leukemic stem cell expansion. The expansion of the NK secretory potential deserves further elucidation. Our study provided new insights into the distribution of JAK2V617F NK functional subsets that may be used as future therapeutic targets for MPN. DisclosuresKobayashi:Pfizer: Consultancy.

PARP inhibitor olaparib has a potential to increase the effectiveness of electrochemotherapy in BRCA1 mutated breast cancer in mice

Electrochemotherapy (ECT), a local therapy, has different effectiveness among tumor types. In breast cancer, its effectiveness is low; therefore, combined therapies are needed. The aim of our study was to combine ECT with PARP inhibitor olaparib, which could inhibit the repair of bleomycin or cisplatin induced DNA damage and potentiate the effectiveness of ECT. The effects of combined therapy were studied in BRCA1 mutated (HCC1937) and non-mutated (HCC1143) triple negative breast cancer cell lines. Therapeutic effectiveness was studied in 2D and 3D cell cultures and in vivo on subcutaneous HCC1937 tumor model in mice. The underlying mechanism of combined therapy was determined with the evaluation of γH2AX foci. Combined therapy of ECT with bleomycin and olaparib potentiated the effectiveness of ECT in BRCA1 mutated HCC1937, but not in non-mutated HCC1143 cells. The combined therapy had a synergistic effect, which was due to the increased number of DNA double strand breaks. Addition of olaparib to ECT with bleomycin in vivo in HCC1937 tumor model had only minimal effect, indicating repetitive olaparib treatment would be needed. This study demonstrates that DNA repair inhibiting drugs, like olaparib, have the potential to increase the effectiveness of ECT with bleomycin.

RET mutated C-cells proliferate more rapidly than non-mutated neoplastic cells.

A statistically significant higher prevalence of the RET p.Met918Thr somatic mutation, identified by direct sequencing, was previously reported in MTC > 2 cm than in smaller tumors. Aim of this study was to correlate the full RET and RAS mutation profile, identified by a Next Generation Sequencing approach, with the growth rate, proliferation and tumor size of MTC. Data of 149 sporadic MTC patients were correlated with RET mutations and Ki67 positivity. Eighty-one cases had a somatic RET mutation, 40 had a RAS mutation and 28 were negative. A statistically significant higher prevalence of RET mutations was found in MTC > 2 cm. A higher prevalence of RET more aggressive mutations, higher allelic frequencies and, higher percentage of Ki67 positive cells were found in larger tumors which had also a worse outcome. Our study highlights the predominant role of RET somatic mutations in MTC tumorigenesis. We demonstrate that RET mutation prevalence and allelic frequency (AF) are significantly higher in larger tumors. Based on these results, we can conclude that RET mutated C-cells’s growth and proliferation are more rapid than those of non-mutated cells and give origin to bigger and more aggressive MTC.

Tumor Microenvironment as a Regulator of Radiation Therapy: New Insights into Stromal-Mediated Radioresistance.

Simple SummaryCancer is multifaceted and consists of more than just a collection of mutated cells. These cancerous cells reside along with other non-mutated cells in an extracellular matrix which together make up the tumor microenvironment or tumor stroma. The composition of the tumor microenvironment plays an integral role in cancer initiation, progression, and response to treatments. In this review, we discuss how the tumor microenvironment regulates the response and resistance to radiation therapy and what targeted agents have been used to combat stromal-mediated radiation resistance.A tumor is a complex “organ” composed of malignant cancer cells harboring genetic aberrations surrounded by a stroma comprised of non-malignant cells and an extracellular matrix. Considerable evidence has demonstrated that components of the genetically “normal” tumor stroma contribute to tumor progression and resistance to a wide array of treatment modalities, including radiotherapy. Cancer-associated fibroblasts can promote radioresistance through their secreted factors, contact-mediated signaling, downstream pro-survival signaling pathways, immunomodulatory effects, and cancer stem cell-generating role. The extracellular matrix can govern radiation responsiveness by influencing oxygen availability and controlling the stability and bioavailability of growth factors and cytokines. Immune status regarding the presence of pro- and anti-tumor immune cells can regulate how tumors respond to radiation therapy. Furthermore, stromal cells including endothelial cells and adipocytes can modulate radiosensitivity through their roles in angiogenesis and vasculogenesis, and their secreted adipokines, respectively. Thus, to successfully eradicate cancers, it is important to consider how tumor stroma components interact with and regulate the response to radiation. Detailed knowledge of these interactions will help build a preclinical rationale to support the use of stromal-targeting agents in combination with radiotherapy to increase radiosensitivity.

Bone Marrow Mesenchymal Stem Cells Support Acute Myeloid Leukemia Bioenergetics and Enhance Antioxidant Defense and Escape from Chemotherapy

SummaryLike normal hematopoietic stem cells, leukemic stem cells depend on their bone marrow (BM) microenvironment for survival, but the underlying mechanisms remain largely unknown. We have studied the contribution of nestin+ BM mesenchymal stem cells (BMSCs) to MLL-AF9-driven acute myeloid leukemia (AML) development and chemoresistance in vivo. Unlike bulk stroma, nestin+ BMSC numbers are not reduced in AML, but their function changes to support AML cells, at the expense of non-mutated hematopoietic stem cells (HSCs). Nestin+ cell depletion delays leukemogenesis in primary AML mice and selectively decreases AML, but not normal, cells in chimeric mice. Nestin+ BMSCs support survival and chemotherapy relapse of AML through increased oxidative phosphorylation, tricarboxylic acid (TCA) cycle activity, and glutathione (GSH)-mediated antioxidant defense. Therefore, AML cells co-opt energy sources and antioxidant defense mechanisms from BMSCs to survive chemotherapy.

Abstract B10: Evaluating YAP and TAZ as therapeutic targets for treating malignant mesothelioma with Hippo pathway disruptions

Abstract Malignant mesotheliomas (MMs) are tumors originating from the mesothelial lining of internal organs. Hippo pathway disruptions are common in MMs; up to 50% of cases exhibit NF2 deficiency and other pathway genes such as LATS2 are also mutated (1). This suggests hyperactivity of YAP and TAZ as a likely oncogenic driver in MMs and a potential axis to exploit therapeutically (2). Before committing to such an approach, a dependence of MMs on YAP/TAZ for survival needs to be tested. We investigated this using different MM cell line models cultured in vitro. The impact of Hippo pathway mutations on YAP and TAZ activity in mesothelial cells was assessed using an NF2 mutant isogenic system in an immortalized mesothelial cell line (Met5A.) YAP activation levels were probed through immunoblotting for YAP S127 phosphorylation, immunofluorescence, and RNA sequencing (RNAseq). YAP and TAZ were inhibited through RNAi-mediated depletion or treatment with simvastatin or verteporfin in a panel of 7 MM cell lines, including 3 with mutations in known Hippo pathway genes and 4 lacking such mutations, and cell viability was measured using an Alamar Blue assay. We found that Hippo pathway-mediated phosphorylation of YAP at S127, and YAP cytoplasmic abundance, was reduced in NF2 mutant Met5A cells compared to isogenic nonmutant cells. Consistent with this, RNAseq analysis showed elevated YAP activity in NF2 mutant Met5A cells. However, RNAi-mediated depletion of YAP and TAZ in a panel of mesothelioma cell lines did not reveal a clear correlation between dependence on YAP/TAZ for survival and aberrant Hippo signaling. There was also a comparable level of sensitivity of NF2 wild-type and NF2 mutant Met5A cells to YAP/TAZ knockdown. Additionally, treatment with verteporfin and simvastatin did not show increased toxicity in cell lines with Hippo pathway mutations relative to nonmutant cells. Our findings confirm that Hippo pathway mutations drive YAP/TAZ hyperactivity in mesothelial cells. However, they show that pathway mutation status does not always correlate with a dependence on YAP and TAZ for mesothelioma cell survival, at least when cultured in vitro. Clearly, a more in-depth analysis of the Hippo pathway in MMs is required to determine the potential of this pathway as a therapeutic target for this disease.

Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes.

Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0–20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.

Proteomic profiling of FBXW7-mutant serous endometrial cancer cells reveals upregulation of PADI2, a potential therapeutic target.

BackgroundDespite advancements over the past decade revealing molecular aberrations characteristic of endometrial cancer (EC) subtypes, serous ECs remain difficult to treat and associated with poor outcomes. This is due, in part, to the rarity of these tumors within clinical trials and the inability to directly target the most frequent genomic abnormalities. One of the most commonly somatically mutated genes in serous ECs is the tumor suppressor F‐box and WD repeat domain containing 7 (FBXW7).MethodsTo identify changes in protein expression associated with FBXW7 mutation, we clustered regularly interspaced short palindromic repeats (CRISPR)‐edited ARK4 FBXW7 nonmutant serous EC cells to insert recurrent FBXW7 mutations. We then compared the liquid chromatography tandem mass spectrometry‐based proteomic profiles of CRISPR‐edited ARK1 and ARK4 serous EC cells to matched parental cells.ResultsAmong distinct total and phosphorylated proteins that were significantly differentially expressed in FBXW7‐mutant cell lines compared to matched parental lines, we identified increased PADI2 (peptidyl arginine deiminase 2) expression in all ARK1 and ARK4 CRISPR‐edited FBXW7‐mutant cell lines. We further confirmed the correlation between FBXW7 mutation and increased PADI2 expression in a third biological background, JHUEM‐1 endometrioid EC cells. Finally, we established that PADI2 protein is expressed in primary serous endometrial tumors.ConclusionOur findings provide novel insight into proteomic changes associated with FBXW7 mutation in serous ECs and identify PADI2 as a novel potential therapeutic target for these tumors.

Efficient Generation of CRISPR/Cas9-Mediated Homozygous/Biallelic Medicago truncatula Mutants Using a Hairy Root System.

In the process of acquiring mutants mediated by CRISPR/Cas9, plantlets are often regenerated from both mutated and non-mutated cells in a random manner, which increase the odds of chimeric mutated plant. In general, it’s necessary to infect more explants or grow to next generation for the need of generating more biallelic or homozygous mutants. In present study, an efficient way of obtaining biallelic or homozygous mutated lines via fast-growing hairy root system without increasing numbers of infected explants or prolonging sexual propagation generation is reported. The fast growing lateral branches of hair roots are originated deep within the parental root from a small number of founder cells at the periphery, and therefore were employed as a library that classify different editing types in different lateral branches in which the homozygous or biallelic lines were screened. Here, MtPDS was employed in a proof-of-concept experiment to evaluate the efficiency of genome editing with our hairy root system. Homozygous/biallelic mutations were found only 1 of the 20 lines in the 1st generation hairy roots, and 8 lines randomly selected were cultured to obtain their branch roots, homozygous/biallelic mutations were found in 6 of the 8 lines in their branch roots. We also tested the method with MtCOMT gene and got the same result. All of the seedlings regenerated from the homozygous/biallelic hairy root mutation lines of MtPDS displayed albino phenotypes. The entire process from vector design to the recovery of plantlets with homozygous/biallelic mutations took approximately 4.5–6.5 months. The whole process could bring inspiration for efficiently generating homozygous/biallelic mutants through CRISPR/Cas9 system from the hairy root or root system of a chimeric mutated transformants, especially for the rare and endangered plants whose explants sources are very limited or the plants that lack of tissue culture and rapid propagation system.

Making sense of giant cell lesions of the jaws (GCLJ): lessons learned from next-generation sequencing.

Next-generation sequencing has revealed mutations in several bone-related lesions and was recently used to uncover the genetic basis of giant cell lesions of the jaws (GCLJ). Consistent with their benign nature, GCLJ show a low tumor mutation burden. They also harbor somatic, heterozygous, mutually exclusive mutations in TRPV4, KRAS, or FGFR1. These signature mutations occur only in a subset of lesional cells, suggesting the existence of a 'landscaping effect', with mutant cells inducing abnormal accumulation of non-mutant cells that form the tumor mass. Osteoclast-rich lesions with histological similarities to GCLJ can occur in the jaws sporadically or in association with genetically inherited syndromes. Based on recent results, the pathogenesis of a subgroup of sporadic GCLJ seems closely related to non-ossifying fibroma of long bones, with both lesions sharing MAPK pathway-activating mutations. In this review, we extrapolate from these recent findings to contextualize GCLJ genetics and we highlight the therapeutic implications of this new information. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Unravelling Heterogeneity of Stem and Progenitor Cells in Myeloid Neoplasms Through Single Cell Multi-omics

Intratumoural heterogeneity (ITH) underlies many of the challenges we face in cancer medicine, including therapy-resistance, disease progression/evolution and relapse after seemingly effective therapy. Distinct tumour cell subpopulations selectively evade therapy and drive disease-progression and technologies that reveal key aspects of ITH are therefore critical for the application of precision cancer medicine. Although bulk genomic analysis has without question provided many insights into genetic ITH, this approach faces a number of fundamental limitations: ITH in cancer occurs at many levels, not restricted to genetics (mutations) but also other factors, such as presence of cancer stem cells in some tumours. Furthermore, bulk genomic analysis reveals patterns of somatic mutations, but not their molecular consequences within distinct (and therapy-resistant) cancer subclones. Whilst many of the scientific questions relating to ITH have remained the same over many decades, our ability to address these questions has advanced dramatically not least because of advances in technology. Ultimately, as the unit of evolution and clonal selection by therapy in cancer is the cell, techniques that resolve heterogeneity at the single-cell level are ideally placed to unravel ITH and provide entirely new insights into cancer biology, with enormous potential to accelerate the development of new approaches to improve outcomes for patients. However, the lack of coverage across key mutation hotspots when studying cancers using single-cell RNA-sequencing techniques has precluded the correlation of genetic and transcriptional readouts from the same single cell, limiting their application to the study of tumors. To overcome such limitation, we developed TARGET-seq, a single cell multi-omic method for the high-sensitivity detection of mutations within single cells in parallel with whole transcriptome analysis. TARGET-seq achieved extremely low allelic dropout rates, allowing resolution of clonal hierarchies with over 98% accuracy, while obtaining unbiased high quality transcriptomes from the same single cell. We have applied TARGET-seq to the study of over ten thousand haematopoietic stem and progenitor cells (HSPCs) from JAK2-mutant myeloproliferative neoplasms. This analysis revealed a high degree of genetic heterogeneity, identifying both linear and branching patterns of clonal evolution. At the transcriptome level different genetic subclones showed distinct transcriptional signatures, indicating that each of them was molecularly distinct. Wild-type cells from MPN patients also showed disrupted gene expression as compared to cells from normal donors, upregulating molecular pathways associated with inflammation (TNFα, TGFβ and IFN signalling). This suggests cell-extrinsic effects disrupting gene expression in non-mutant cells, which has been shown to have prognostic significance and might underlie therapy response. Moreover, TARGET-seq analysis allowed us to identify putative biomarkers of JAK2V617F mutant cells, including novel therapeutic targets to selectively eradicate JAK2-mutant cells and importantly, potential candidates for antibody-based immunotherapy. Analysis of samples from MPN patients undergoing disease transformation to Acute Myeloid Leukemia (sAML) revealed striking patterns of clonal evolution in different immunophenotypically-defined cell types. We identified pre-leukemic and leukemic subclones emerging from hematopoietic stem cells rather than more mature progenitors, in contrast to evolution patterns in de novo AML, which might indicate different cancer stem cell reservoirs. In summary, TARGET-seq allowed us to identify distinct and biologically relevant molecular signatures of different genetic subclones of HSPCs in myeloproliferative neoplasms. TARGET-seq could also be broadly applied to the study of other types of tumours, providing a powerful tool for biomarker and therapeutic target discovery for precision medicine. Disclosures Mead: Bristol Myers-Squibb: Consultancy; Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel/accommodation expenses, Research Funding, Speakers Bureau; CTI: Honoraria, Research Funding; Celgene: Consultancy, Research Funding.