Time-course RNAseq data of murine AB1 mesothelioma and Renca renal cancer following immune checkpoint therapy.

Time-critical transcriptional events in the immune microenvironment are important for response to immune checkpoint blockade (ICB), yet these events are difficult to characterise and remain incompletely understood. Here, we present whole tumor RNA sequencing data in the context of treatment with ICB in murine models of AB1 mesothelioma and Renca renal cell cancer. We sequenced 144 bulk RNAseq samples from these two cancer types across 4 time points prior and after treatment with ICB. We also performed single-cell sequencing on 12 samples of AB1 and Renca tumors an hour before ICB administration. Our samples were equally distributed between responders and non-responders to treatment. Additionally, we sequenced AB1-HA mesothelioma tumors treated with two sample dissociation protocols to assess the impact of these protocols on the quality transcriptional information in our samples. These datasets provide time-course information to transcriptionally characterize the ICB response and provide detailed information at the single-cell level of the early tumor microenvironment prior to ICB therapy.

Transcription factor 3 is dysregulated in megakaryocytes in myelofibrosis.

Transcription factor 3 (TCF3) is a DNA transcription factor that modulates megakaryocyte development. Although abnormal TCF3 expression has been identified in a range of hematological malignancies, to date, it has not been investigated in myelofibrosis (MF). MF is a Philadelphia-negative myeloproliferative neoplasm (MPN) that can arise de novo or progress from essential thrombocythemia [ET] and polycythemia vera [PV] and where dysfunctional megakaryocytes have a role in driving the fibrotic progression. We aimed to examine whether TCF3 is dysregulated in megakaryocytes in MPN, and specifically in MF. We first assessed TCF3 protein expression in megakaryocytes using an immunohistochemical approach analyses and showed that TCF3 was reduced in MF compared with ET and PV. Further, the TCF3-negative megakaryocytes were primarily located near trabecular bone and had the typical "MF-like" morphology as described by the WHO. Genomic analysis of isolated megakaryocytes showed three mutations, all predicted to result in a loss of function, in patients with MF; none were seen in megakaryocytes isolated from ET or PV marrow samples. We then progressed to transcriptomic sequencing of platelets which showed loss of TCF3 in MF. These proteomic, genomic and transcriptomic analyses appear to indicate that TCF3 is downregulated in megakaryocytes in MF. This infers aberrations in megakaryopoiesis occur in this progressive phase of MPN. Further exploration of this pathway could provide insights into TCF3 and the evolution of fibrosis and potentially lead to new preventative therapeutic targets.

What is the context? We investigated TCF3 (transcription factor 3), a gene that regulates megakaryocyte development, for genomic and proteomic changes in myelofibrosis.Myelofibrosis is the aggressive phase of a group of blood cancers called myeloproliferative neoplasms, and abnormalities in development and maturation of megakaryocytes is thought to drive the development of myelofibrosis.What is new? We report detection of three novel TCF3 mutations in megakaryocytes and decreases in TCF3 protein and gene expression in primary megakaryocytes and platelets from patients with myelofibrosis.This is the first association between loss of TCF3 in megakaryocytes from patients and myelofibrosis.What is the impact? TCF3 dysregulation may be a novel mechanism that is responsible for the development of myelofibrosis and better understanding of this pathway could identify new drug targets.

PlateletSeq: A novel method for discovery of blood-based biomarkers.

Platelets are small circulating fragments of cells that play important roles in thrombosis, haemostasis, immune response, inflammation and cancer growth. Although anucleate, they contain a rich RNA repertoire which offers an opportunity to characterise changes in platelet gene expression in health and disease. Whilst this can be achieved with conventional RNA sequencing, a large input of high-quality RNA, and hence blood volume, is required (unless a pre-amplification step is added), along with specialist bioinformatic skills for data analysis and interpretation. We have developed a transcriptomics next-generation sequencing-based approach that overcomes these limitations. Termed PlateletSeq, this method requires very low levels of RNA input and does not require specialist bioinformatic analytical skills. Here we describe the methodology, from sample collection to processing and data analysis. Specifically, blood samples can be stored for up to 8 days at 4 °C prior to analysis. Platelets are isolated using multi-step centrifugation and a purity of ≤ 1 leucocyte per 0.26x106 platelets is optimal for gene expression analysis. We have applied PlateletSeq to normal adult blood samples and show there are no age-associated variations and only minor gender-associated differences. In contrast, platelets from patients with myeloproliferative neoplasms show differences in platelet transcript profiles from normal and between disease subtypes. This illustrates the potential applicability of PlateletSeq for biomarker discovery and studying platelet biology in patient samples. It also opens avenues for assessing platelet quality in other fields such as transfusion research.

Temporally restricted activation of IFNß signaling underlies response to immune checkpoint therapy in mice.

The biological determinants of the response to immune checkpoint blockade (ICB) in cancer remain incompletely understood. Little is known about dynamic biological events that underpin therapeutic efficacy due to the inability to frequently sample tumours in patients. Here, we map the transcriptional profiles of 144 responding and non-responding tumours within two mouse models at four time points during ICB. We find that responding tumours display on/fast-off kinetics of type-I-interferon (IFN) signaling. Phenocopying of this kinetics using time-dependent sequential dosing of recombinant IFNs and neutralizing antibodies markedly improves ICB efficacy, but only when IFNß is targeted, not IFNα. We identify Ly6C+/CD11b+ inflammatory monocytes as the primary source of IFNß and find that active type-I-IFN signaling in tumour-infiltrating inflammatory monocytes is associated with T cell expansion in patients treated with ICB. Together, our results suggest that on/fast-off modulation of IFNß signaling is critical to the therapeutic response to ICB, which can be exploited to drive clinical outcomes towards response.

The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production.

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine-rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

Mutation profile of acute myeloid leukaemia in a Chinese cohort by targeted next-generation sequencing.

BACKGROUND: Acute myeloid leukaemia (AML) results from the clonal expansion of blast cells of myeloid origin driven by genomic defects. The advances in next-generation sequencing (NGS) have allowed the identification of many mutated genes important in the pathogenesis of AML. AIMS: In this study, we aimed to assess the mutation types and frequency in a Chinese cohort presenting with de novo AML cohort using a targeted NGS strategy. METHODS: In total, we studied samples from 87 adult patients with de novo AML who had no prior history of cytotoxic chemotherapy. Samples were evaluated using a 120-gene targeted NGS panel to assess the mutation profile. RESULTS: Of the 87 AML patients, there were 60 (69%) with a normal karyotype. 89.7% of patients had variants, with an average of 1.9 mutations per patient (range: 0-5 mutations per patient). DNMT3A variants were the most common, being detected in 33 patients (37.9%). NPM1 (34.5%), IDH1/2 (24.1%) and FLT3-ITD (20.7%) mutations was the next most common. Of the patients with DNMT3A mutations, 24.2% also had mutations NPM1 and FLT3-ITD and 6.1% NPM1, FLT3-ITD and IDH mutations. CONCLUSION: Both DNMT3A and NPM1 mutations were more common than in other Chinese and Western AML cohorts that have been studied. DNMT3A mutations tended to co-occur with NPM1 and FLT3-ITD mutations and were most commonly seen with a normal karyotype.

Myeloid somatic mutation panel testing in myeloproliferative neoplasms.

Myeloproliferative neoplasms are characterised by somatic mutations in pathways that regulate cell proliferation, epigenetic modifications, RNA splicing or DNA repair. Assessment of the mutational profile assists diagnosis and classification, but also aids assessment of prognosis, and may guide the use of emerging targeted therapies. The most practical way to provide information on numerous genetic variants is by using massively parallel sequencing, commonly in the form of disease specific next generation sequencing (NGS) panels. This review summarises the diagnostic and prognostic value of somatic mutation testing in Philadelphia-negative myeloproliferative neoplasms: polycythaemia vera, essential thrombocythaemia, primary myelofibrosis, chronic neutrophilic leukaemia, systemic mastocytosis, and chronic eosinophilic leukaemia. NGS panel testing is increasing in routine practice and promises to improve the accuracy and efficiency of pathological diagnosis and prognosis.

Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows.

BACKGROUND: Single-cell RNA sequencing has been widely adopted to estimate the cellular composition of heterogeneous tissues and obtain transcriptional profiles of individual cells. Multiple approaches for optimal sample dissociation and storage of single cells have been proposed as have single-nuclei profiling methods. What has been lacking is a systematic comparison of their relative biases and benefits. RESULTS: Here, we compare gene expression and cellular composition of single-cell suspensions prepared from adult mouse kidney using two tissue dissociation protocols. For each sample, we also compare fresh cells to cryopreserved and methanol-fixed cells. Lastly, we compare this single-cell data to that generated using three single-nucleus RNA sequencing workflows. Our data confirms prior reports that digestion on ice avoids the stress response observed with 37 °C dissociation. It also reveals cell types more abundant either in the cold or warm dissociations that may represent populations that require gentler or harsher conditions to be released intact. For cell storage, cryopreservation of dissociated cells results in a major loss of epithelial cell types; in contrast, methanol fixation maintains the cellular composition but suffers from ambient RNA leakage. Finally, cell type composition differences are observed between single-cell and single-nucleus RNA sequencing libraries. In particular, we note an underrepresentation of T, B, and NK lymphocytes in the single-nucleus libraries. CONCLUSIONS: Systematic comparison of recovered cell types and their transcriptional profiles across the workflows has highlighted protocol-specific biases and thus enables researchers starting single-cell experiments to make an informed choice.

Platelets in myeloproliferative neoplasms have a distinct transcript signature in the presence of marrow fibrosis.

Marrow fibrosis is a significant complication of myeloproliferative neoplasms (MPN) that affects up to 20% of patients and is associated with a poor prognosis. The pathological processes that lead to fibrotic progression are not well understood, but megakaryocytes have been implicated in the process. The aim of this study was to determine whether platelets, derived from megakaryocytes, have transcriptomic alterations associated with fibrosis. Platelets from MPN patients with and without fibrosis and non-malignant control individuals were assessed using next generation sequencing. Results from the initial training cohort showed discrete changes in platelet transcripts in the presence of marrow fibrosis. We identified more than 1000 differentially expressed transcripts from which a putative 3-gene fibrotic platelet signature (CCND1, H2AX [previously termed H2AFX] and CEP55) could be identified. This fibrosis-associated signature was assessed blinded on platelets from an independent test MPN patient cohort. The 3-gene signature was able to discriminate between patients with and without marrow fibrosis with a positive predictive value of 71% (93% specificity, 71% sensitivity). This demonstrates that assessment of dysregulated transcripts in platelets may be a useful monitoring tool in MPN to identify progression to marrow fibrosis. Further, sequential monitoring could have clinical applications for early prediction of progression to fibrosis.

Automated enumeration of lymphoid and plasma cells in bone marrow to establish normal reference ranges.

AIMS: The number of precursor and mature lymphoid cells and plasma cells in normal bone marrow trephine (BMT) biopsies throughout the human lifespan is unknown. Reference ranges have been established from aspirated marrow, but due to haemodilution errors, these do not accurately reflect the native marrow milieu. We aimed to define age-specific, normal reference ranges for lymphoid and plasma cells in BMT biopsy specimens using a combined immunophenotyping and digital enumeration approach. METHODS: Morphologically normal BMT biopsy specimens (n=483) were obtained from patients aged 1 month to 90 years of age. Immunohistochemistry was performed to identify lymphoid progenitors , T-lymphocytes (CD3), B-lymphocytes (CD20) and plasma cells (CD138 and MUM1). Positive cells were counted using digital enumeration software, and the percent positivity for each antigen was determined per case. Mean values were generated for specific age groups, and age-defined reference ranges were determined for each antigen using normalised data. RESULTS: A mean of 16 609 cells (range: 7210-34 097) were counted per biopsy. Infant marrows showed a predominance of immature lymphoid progenitors and B cells. With increasing age, an increase in mean T cell and plasma cell numbers were observed. The results showed the same trends to flow cytometry references for aspirate material although the absolute values differed. CONCLUSIONS: Combined immunohistochemistry and automated enumeration gives an accurate, reproducible number of antigen-positive cells and has generated normal reference ranges for these cell types in BMT biopsies. The method and ranges we have established have the potential to be applied in routine clinical practice.

Megakaryocytes in Myeloproliferative Neoplasms Have Unique Somatic Mutations.

Myeloproliferative neoplasms (MPNs) are a group of related clonal hemopoietic stem cell disorders associated with hyperproliferation of myeloid cells. They are driven by mutations in the hemopoietic stem cell, most notably JAK2V617F, CALR, and MPL. Clinically, they have the propensity to progress to myelofibrosis and transform to acute myeloid leukemia. Megakaryocytic hyperplasia with abnormal features are characteristic, and it is thought that these cells stimulate and drive fibrotic progression. The biological defects underpinning this remain to be explained. In this study we examined the megakaryocyte genome in 12 patients with MPNs to determine whether there are somatic variants and whether there is any association with marrow fibrosis. We performed targeted next-generation sequencing for 120 genes associated with myeloid neoplasms on megakaryocytes isolated from aspirated bone marrow. Ten of the 12 patients had genomic defects in megakaryocytes that were not present in nonmegakaryocytic hemopoietic marrow cells from the same patient. The greatest allelic burden was in patients with increased reticulin deposition. The megakaryocyte-unique mutations were predominantly in genes that regulate chromatin remodeling, chromosome alignment, and stability. These findings show that genomic abnormalities are present in megakaryocytes in MPNs and that these appear to be associated with progression to bone marrow fibrosis.

Dysregulation of the intrinsic apoptotic pathway mediates megakaryocytic hyperplasia in myeloproliferative neoplasms.

AIMS: Megakaryocyte expansion in myeloproliferative neoplasms (MPNs) is due to uncontrolled proliferation accompanied by dysregulation of proapoptotic and antiapoptotic mechanisms. Here we have investigated the intrinsic and extrinsic apoptotic pathways of megakaryocytes in human MPNs to further define the mechanisms involved. METHODS: The megakaryocytic expression of proapoptotic caspase-8, caspase-9, Diablo, p53 and antiapoptotic survivin proteins was investigated in bone marrow specimens of the MPNs (n=145) and controls (n=15) using immunohistochemistry. The megakaryocyte percentage positivity was assessed by light microscopy and correlated with the MPN entity, JAK2V617F/CALR mutation status and platelet count. RESULTS: The proportion of megakaryocytes in the MPNs expressing caspase-8, caspase-9, Diablo, survivin and p53 was significantly greater than controls. A greater proportion of myeloproliferative megakaryocytes expressed survivin relative to its reciprocal inhibitor, Diablo. Differences were seen between myelofibrosis, polycythaemia vera and essential thrombocythaemia for caspase-9 and p53. CALR-mutated cases had greater megakaryocyte p53 positivity compared to those with the JAK2V617F mutation. Proapoptotic caspase-9 expression showed a positive correlation with platelet count, which was most marked in myelofibrosis and CALR-mutated cases. CONCLUSIONS: Disruptions targeting the intrinsic apoptotic cascade promote megakaryocyte hyperplasia and thrombocytosis in the MPNs. There is progressive dysfunction of apoptosis as evidenced by the marked reduction in proapoptotic caspase-9 and accumulation of p53 in myelofibrosis. The dysfunction of caspase-9, which is necessary for proplatelet formation, may be the mechanism for the excess thrombocytosis associated with CALR mutations. Survivin seems to be the key protein mediating the megakaryocyte survival signature in the MPNs and is a potential therapeutic target.

Stimulating the Release of Exosomes Increases the Intercellular Transfer of Prions.

Exosomes are small extracellular vesicles released by cells and play important roles in intercellular communication and pathogen transfer. Exosomes have been implicated in several neurodegenerative diseases, including prion disease and Alzheimer disease. Prion disease arises upon misfolding of the normal cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). The disease has a unique transmissible etiology, and exosomes represent a novel and efficient method for prion transmission. The precise mechanism by which prions are transmitted from cell to cell remains to be fully elucidated, although three hypotheses have been proposed: direct cell-cell contact, tunneling nanotubes, and exosomes. Given the reported presence of exosomes in biological fluids and in the lipid and nucleic acid contents of exosomes, these vesicles represent an ideal mechanism for encapsulating prions and potential cofactors to facilitate prion transmission. This study investigates the relationship between exosome release and intercellular prion dissemination. Stimulation of exosome release through treatment with an ionophore, monensin, revealed a corresponding increase in intercellular transfer of prion infectivity. Conversely, inhibition of exosome release using GW4869 to target the neutral sphingomyelinase pathway induced a decrease in intercellular prion transmission. Further examination of the effect of monensin on PrP conversion revealed that monensin also alters the conformational stability of PrP(C), leading to increased generation of proteinase K-resistant prion protein. The findings presented here provide support for a positive relationship between exosome release and intercellular transfer of prion infectivity, highlighting an integral role for exosomes in facilitating the unique transmissible nature of prions.

Megakaryocytic hyperplasia in myeloproliferative neoplasms is driven by disordered proliferative, apoptotic and epigenetic mechanisms.

AIMS: Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal proliferative bone marrow diseases characterised by extensive megakaryocytic hyperplasia and morphological atypia. Despite knowledge of genomic defects, the pathobiological processes driving these megakaryocytic abnormalities in MPN remain poorly explained. We have explored the proliferative, apoptotic and epigenetic profiles of megakaryocytes in human MPN. METHODS: Immunohistochemical staining was performed on bone marrow trephine biopsies of 81 MPN (with and without JAK2(V617F) and CALR mutations) and 15 normal controls to assess the megakaryocytic expression of biomarkers associated with proliferation (Ki67), apoptosis (Bcl-XL, BNIP-3) and epigenetic regulation (EZH2, SUZ12). RESULTS: Myeloproliferative megakaryocytes showed significantly greater expression of proliferative Ki67 and anti-apoptotic Bcl-XL, reduced pro-apoptotic BNIP-3 and increased SUZ12 compared with controls. In essential thrombocythaemia, large-giant megakaryocytes with hyperlobated nuclei showed a trend towards a proliferative signature. In contrast, myelofibrotic megakaryocytes with condensed nuclear chromatin, and cases with CALR mutations, had significant reductions in pro-apoptotic BNIP-3. CONCLUSIONS: Uncontrolled megakaryocytic expansion in MPN results from a combination of increased proliferation, attenuated apoptosis and defective epigenetic regulation with CALR mutations favouring apoptotic failure. The higher platelet counts reported to be seen in MPN with CALR mutations may be due to greater dysregulation of megakaryocyte apoptosis.

The secret life of extracellular vesicles in metal homeostasis and neurodegeneration.

Biologically active metals such as copper, zinc and iron are fundamental for sustaining life in different organisms with the regulation of cellular metal homeostasis tightly controlled through proteins that coordinate metal uptake, efflux and detoxification. Many of the proteins involved in either uptake or efflux of metals are localised and function on the plasma membrane, traffic between intracellular compartments depending upon the cellular metal environment and can undergo recycling via the endosomal pathway. The biogenesis of exosomes also occurs within the endosomal system, with several major neurodegenerative disease proteins shown to be released in association with these vesicles, including the amyloid-ß (Aß) peptide in Alzheimer's disease and the infectious prion protein involved in Prion diseases. Aß peptide and the prion protein also bind biologically active metals and are postulated to play important roles in metal homeostasis. In this review, we will discuss the role of extracellular vesicles in Alzheimer's and Prion diseases and explore their potential contribution to metal homeostasis.

The neutral sphingomyelinase pathway regulates packaging of the prion protein into exosomes.

Prion diseases are a group of transmissible, fatal neurodegenerative disorders associated with the misfolding of the host-encoded prion protein, PrP(C), into a disease-associated form, PrP(Sc). The transmissible prion agent is principally formed of PrP(Sc) itself and is associated with extracellular vesicles known as exosomes. Exosomes are released from cells both in vitro and in vivo, and have been proposed as a mechanism by which prions spread intercellularly. The biogenesis of exosomes occurs within the endosomal system, through formation of intraluminal vesicles (ILVs), which are subsequently released from cells as exosomes. ILV formation is known to be regulated by the endosomal sorting complexes required for transport (ESCRT) machinery, although an alternative neutral sphingomyelinase (nSMase) pathway has been suggested to also regulate this process. Here, we investigate a role for the nSMase pathway in exosome biogenesis and packaging of PrP into these vesicles. Inhibition of the nSMase pathway using GW4869 revealed a role for the nSMase pathway in both exosome formation and PrP packaging. In agreement, targeted knockdown of nSMase1 and nSMase2 in mouse neurons using lentivirus-mediated RNAi also decreases exosome release, demonstrating the nSMase pathway regulates the biogenesis and release of exosomes. We also demonstrate that PrP(C) packaging is dependent on nSMase2, whereas the packaging of disease-associated PrP(Sc) into exosomes occurs independently of nSMase2. These findings provide further insight into prion transmission and identify a pathway which directly assists exosome-mediated transmission of prions.

Prion infection impairs cholesterol metabolism in neuronal cells.

Conversion of prion protein (PrP(C)) into a pathological isoform (PrP(Sc)) during prion infection occurs in lipid rafts and is dependent on cholesterol. Here, we show that prion infection increases the abundance of cholesterol transporter, ATP-binding cassette transporter type A1 (ATP-binding cassette transporter type A1), but reduces cholesterol efflux from neuronal cells leading to the accumulation of cellular cholesterol. Increased abundance of ABCA1 in prion disease was confirmed in prion-infected mice. Mechanistically, conversion of PrP(C) to the pathological isoform led to PrP(Sc) accumulation in rafts, displacement of ABCA1 from rafts and the cell surface, and enhanced internalization of ABCA1. These effects were abolished with reversal of prion infection or by loading cells with cholesterol. Stimulation of ABCA1 expression with liver X receptor agonist or overexpression of heterologous ABCA1 reduced the conversion of prion protein into the pathological form upon infection. These findings demonstrate a reciprocal connection between prion infection and cellular cholesterol metabolism, which plays an important role in the pathogenesis of prion infection in neuronal cells.

Pathogenic mutations within the hydrophobic domain of the prion protein lead to the formation of protease-sensitive prion species with increased lethality.

UNLABELLED: Prion diseases are a group of fatal and incurable neurodegenerative diseases affecting both humans and animals. The principal mechanism of these diseases involves the misfolding the host-encoded cellular prion protein, PrP(C), into the disease-associated isoform, PrP(Sc). Familial forms of human prion disease include those associated with the mutations G114V and A117V, which lie in the hydrophobic domain of PrP. Here we have studied the murine homologues (G113V and A116V) of these mutations using cell-based and animal models of prion infection. Under normal circumstances, the mutant forms of PrP(C) share similar processing, cellular localization, and physicochemical properties with wild-type mouse PrP (MoPrP). However, upon exposure of susceptible cell lines expressing these mutants to infectious prions, very low levels of protease-resistant aggregated PrP(Sc) are formed. Subsequent mouse bioassay revealed high levels of infectivity present in these cells. Thus, these mutations appear to limit the formation of aggregated PrP(Sc), giving rise to the accumulation of a relatively soluble, protease sensitive, prion species that is highly neurotoxic. Given that these mutations lie next to the glycine-rich region of PrP that can abrogate prion infection, these findings provide further support for small, protease-sensitive prion species having a significant role in the progression of prion disease and that the hydrophobic domain is an important determinant of PrP conversion. IMPORTANCE: Prion diseases are transmissible neurodegenerative diseases associated with an infectious agent called a prion. Prions are comprised of an abnormally folded form of the prion protein (PrP) that is normally resistant to enzymes called proteases. In humans, prion disease can occur in individuals who inherited mutations in the prion protein gene. Here we have studied the effects of two of these mutations and show that they influence the properties of the prions that can be formed. We show that the mutants make highly infectious prions that are more sensitive to protease treatment. This study highlights a certain region of the prion protein as being involved in this effect and demonstrates that prions are not always resistant to protease treatment.