Comparative Analyses of Targeted Myeloid Cancer Next-Generation Sequencing Panel in Fresh Blood, Bone Marrow and FFPE Material.

Next-generation sequencing is a vital tool for personalized diagnostics and therapies in cancer. Despite numerous advantages, the method depends on multiple parameters regarding the sample material, e.g., sample fixation. A panel's ability to ensure balanced pre-amplification of the regions of interest is challenging, especially in targeted sequencing approaches, but of significant importance to its applicability across hematological malignancies and solid tumors. This study comparatively evaluated the technical performance of the commercially available OncomineTM Myeloid Panel in fresh and Formalin-fixed paraffin-embedded (FFPE) material by using an Ion Torrent™ Personal Genome Machine™ System and Ion GeneStudio S5 System platform. In total, 114 samples were analyzed, including 55 fresh materials and 59 FFPE samples. Samples were sequenced with a minimum of one million reads. Amplicons with coverage below 400 reads were classified as underperforming. In fresh material, 49/526 amplicons were identified as performing insufficiently, corresponding with 18 genes. Using FFPE material, 103/526 amplicons underperformed. Independent of input material, regions in 27 genes, including ASXL1, BCOR and BRAF, did not match quality parameters. Subsequently, exemplary mutations were extracted from the Catalogue of Somatic Mutations in Cancer database. This technical evaluation of the OncomineTM Myeloid Panel identified amplicons that do not achieve adequate coverage levels and which need to be considered when interpreting sequencing.

Combined BCL-2 and PI3K/AKT Pathway Inhibition in KMT2A-Rearranged Acute B-Lymphoblastic Leukemia Cells.

Numerous hematologic neoplasms, including acute B-lymphoblastic leukemia (B-ALL), are characterized by overexpression of anti-apoptotic BCL-2 family proteins. Despite the high clinical efficacy of the specific BCL-2 inhibitor venetoclax in acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL), dose limitation and resistance argue for the early exploration of rational combination strategies. Recent data indicated that BCL-2 inhibition in B-ALL with KMT2A rearrangements is a promising intervention option; however, combinatorial approaches have not been in focus so far. The PI3K/AKT pathway has emerged as a possible target structure due to multiple interactions with the apoptosis cascade as well as relevant dysregulation in B-ALL. Herein, we demonstrate for the first time that combined BCL-2 and PI3K/AKT inhibition has synergistic anti-proliferative effects on B-ALL cell lines. Of note, all tested combinations (venetoclax + PI3K inhibitors idelalisib or BKM-120, as well as AKT inhibitors MK-2206 or perifosine) achieved comparable anti-leukemic effects. In a detailed analysis of apoptotic processes, among the PI3K/AKT inhibitors only perifosine resulted in an increased rate of apoptotic cells. Furthermore, the combination of venetoclax and perifosine synergistically enhanced the activity of the intrinsic apoptosis pathway. Subsequent gene expression studies identified the pro-apoptotic gene BBC3 as a possible player in synergistic action. All combinatorial approaches additionally modulated extrinsic apoptosis pathway genes. The present study provides rational combination strategies involving selective BCL-2 and PI3K/AKT inhibition in B-ALL cell lines. Furthermore, we identified a potential mechanistic background of the synergistic activity of combined venetoclax and perifosine application.

Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts.

Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.

Evaluation of Pan-RAF Inhibitor LY3009120 on Human Uveal Melanoma Cell Line 92-1.

BACKGROUND/AIM: Uveal melanoma (UM) represents a prevailing primary intraocular malignancy, with a limited median overall survival among metastatic patients, and most tumors lack RAF/RAS mutations. The pan-RAF inhibitor LY3009120 has demonstrated valuable anti-tumor effects in a wide range of RAF/RASmut and wild-type (WT) tumor models. This study aimed to evaluate the antitumor effect of LY3009120 on 92-1 UM cell line. MATERIALS AND METHODS: The effect of the pan-RAF inhibitor LY3009120 on cell proliferation, metabolic activity, biomass, early and late apoptosis/necrosis, and morphology was characterized in vitro (0.1-5 µM for 48 h/72 h). Furthermore, targeted panel sequencing was used to characterize the mutational landscape of the human 92-1 UM cell line. RESULTS: LY3009120 showed a significant concentration-dependent anti-proliferative effect on 92-1 cells. Cell proliferation and viability were significantly reduced at the lowest effective concentration of 0.5 µM (at 48 and 72 h, p<0.001). Furthermore, LY3009120 caused significant early apoptosis and late apoptosis/necrosis in 92-1 cells at 5 µM. Except for TP53, NGS showed that all 49 additional analysed genes (Oncomine myeloid panel) of 92-1 were wild-type, including BRAF, NRAS, and KRAS. CONCLUSION: The pan-RAF inhibitor LY3009120 demonstrated a significant anti-tumor effect on human UM cell line 92-1 independent of the molecular BRAF and RAS mutational status.

Generation of the human iPSC lines AKOSi011-A carrying the mutation p.Pro65Ser/p.Asp35T and AKOSi012-A, carrying the mutation p.Tyr231His, derived from FAHN patient fibroblasts.

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a hereditary neurodegenerative disease caused by mutations in the FA2H gene. Patients show a wide range of neurological symptoms and an abnormal myelination. Here we describe the generation of the human induced pluripotent stem cell (hiPSC) lines AKOSi011-A and AKOSi012-A, derived from FAHN-patient fibroblasts, carrying the compound heterozygous mutation p.Pro65Ser/p.Asp35Tyr and the homozygous mutation p.Tyr231His, respectively. The hiPSC lines were generated using a non-integrating Sendai virus. The obtained hiPSCs show an unobtrusive karyotype, carry the mutations of the original fibroblasts, express pluripotency markers and can differentiate into cells of the three germ layers.

The TiHoCL panel for canine lymphoma: a feasibility study integrating functional genomics and network biology approaches for comparative oncology targeted NGS panel design.

Targeted next-generation sequencing (NGS) enables the identification of genomic variants in cancer patients with high sensitivity at relatively low costs, and has thus opened the era to personalized human oncology. Veterinary medicine tends to adopt new technologies at a slower pace compared to human medicine due to lower funding, nonetheless it embraces technological advancements over time. Hence, it is reasonable to assume that targeted NGS will be incorporated into routine veterinary practice in the foreseeable future. Many animal diseases have well-researched human counterparts and hence, insights gained from the latter might, in principle, be harnessed to elucidate the former. Here, we present the TiHoCL targeted NGS panel as a proof of concept, exemplifying how functional genomics and network approaches can be effectively used to leverage the wealth of information available for human diseases in the development of targeted sequencing panels for veterinary medicine. Specifically, the TiHoCL targeted NGS panel is a molecular tool for characterizing and stratifying canine lymphoma (CL) patients designed based on human non-Hodgkin lymphoma (NHL) research outputs. While various single nucleotide polymorphisms (SNPs) have been associated with high risk of developing NHL, poor prognosis and resistance to treatment in NHL patients, little is known about the genetics of CL. Thus, the ~100 SNPs featured in the TiHoCL targeted NGS panel were selected using functional genomics and network approaches following a literature and database search that shielded ~500 SNPs associated with, in nearly all cases, human hematologic malignancies. The TiHoCL targeted NGS panel underwent technical validation and preliminary functional assessment by sequencing DNA samples isolated from blood of 29 lymphoma dogs using an Ion Torrent™ PGM System achieving good sequencing run metrics. Our design framework holds new possibilities for the design of similar molecular tools applied to other diseases for which limited knowledge is available and will improve drug target discovery and patient care.

Generation of the human iPSC line AKOSi010-A from fibroblasts of a female FAHN patient, carrying the compound heterozygous mutation p.Gly45Arg/p.His319Arg.

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare childhood onset neurodegenerative disease caused by mutations in the FA2H gene. Patients display abnormal myelination, cerebellar atrophy and some have iron deposition in the central nervous system. Here we describe the generation of AKOSi010-A, a human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a female patient carrying the compound heterozygous p.Gly45Arg/p.His319Arg, using non-integrating Sendai virus. The generated iPSCs express pluripotency markers, can differentiate into cell types of the three germ layers and show a normal karyotype. This cell line displays a unique source to study the pathophysiology of FAHN.

Effective tumor cell abrogation via Venetoclax-mediated BCL-2 inhibition in KMT2A-rearranged acute B-lymphoblastic leukemia.

Dysregulation of the intrinsic BCL-2 pathway-mediated apoptosis cascade is a common feature of hematological malignancies including acute B-lymphoblastic leukemia (B-ALL). The KMT2A-rearranged high-risk cytogenetic subtype is characterized by high expression of antiapoptotic protein BCL-2, likely due to the direct activating binding of KMT2A fusion proteins to the BCL2 gene. The BCL-2 inhibitor venetoclax (VEN) has proven great clinical value in other blood cancers, however, data on B-ALL is sparse and past studies have not so far described the effects of VEN on gene and protein expression profiles. Using cell lines and patient-derived in vivo xenograft models, we show BCL-2 pathway-mediated apoptosis induction and decelerated tumor cell counts in KMT2A-rearranged B-ALL but not in other cytogenetic subtypes. VEN treatment of cell line- and patient-derived xenografts reduced blast frequencies in blood, bone marrow, and spleen, and tumor cell doubling times were increased. Growth rates are further correlated with VEN concentrations in blood. In vitro incubation with VEN resulted in BCL-2 dephosphorylation and targeted panel RNA sequencing revealed reduced gene expression of antiapoptotic pathway members BCL2, MCL1, and BCL2L1 (BCL-XL). Reinforced translocation of BAX proteins towards mitochondria induced caspase activation and cell death commitment. Prolonged VEN application led to upregulation of antiapoptotic proteins BCL-2, MCL-1, and BCL-XL. Interestingly, the extrinsic apoptosis pathway was strongly modulated in SEM cells in response to VEN. Gene expression of members of the tumor necrosis factor signaling cascade was increased, resulting in canonical NF-kB signaling. This possibly suggests a previously undescribed mechanism of BCL-2-independent and NF-kB-mediated upregulation of MCL-1 and BCL-XL. In summary, we herein prove that VEN is a potent option to suppress tumor cells in KMT2A-rearranged B-ALL in vitro and in vivo. Possible evasion mechanisms, however, must be considered in subsequent studies.

The Molecular Subtype of Adult Acute Lymphoblastic Leukemia Samples Determines the Engraftment Site and Proliferation Kinetics in Patient-Derived Xenograft Models.

In acute lymphoblastic leukemia (ALL), conventional cell lines do not recapitulate the clonal diversity and microenvironment. Orthotopic patient-derived xenograft models (PDX) overcome these limitations and mimic the clinical situation, but molecular stability and engraftment patterns have not yet been thoroughly assessed. We herein describe and characterize the PDX generation in NSG mice. In vivo tumor cell proliferation, engraftment and location were monitored by flow cytometry and bioluminescence imaging. Leukemic cells were retransplanted for up to four passages, and comparative analyses of engraftment pattern, cellular morphology and genomic hotspot mutations were conducted. Ninety-four percent of all samples were successfully engrafted, and the xenograft velocity was dependent on the molecular subtype, outcome of the patient and transplantation passage. While BCR::ABL1 blasts were located in the spleen, KMT2A-positive cases had higher frequencies in the bone marrow. Molecular changes appeared in most model systems, with low allele frequency variants lost during primary engraftment. After the initial xenografting, however, the PDX models demonstrated high molecular stability. This protocol for reliable ALL engraftment demonstrates variability in the location and molecular signatures during serial transplantation. Thorough characterization of experimentally used PDX systems is indispensable for the correct analysis and valid data interpretation of preclinical PDX studies.