Self-renewal of single mouse hematopoietic stem cells is reduced by JAK2V617F without compromising progenitor cell expansion

Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers

The acute side effects of d-amphetamine and methamphetamine on simulated driving performance, cognitive functioning, brain activity, and the standardised field sobriety tests

Recently there has been an increase in awareness of the role of drugs other than alcohol in the causation of road accidents and deaths, with the most recent report indicating that 33% of all Victorian (Australia) road fatalities are drug (other than alcohol) related (TAC, 2006). Currently in Victoria, one of the classes of drugs reported to be of most concern is the amphetamines. The epidemiological driving literature highlights a possible association between amphetamine use and road crashes. However, since the cognitive research generally indicates cognitive enhancing properties following amphetamine consumption, it remains unclear how amphetamines may be related to adverse driving. The present thesis was designed to explore this issue. In response to the increasing number of drug-related road fatalities, the Standardised Field Sobriety Tests (SFSTs), designed and validated for the detection and assessment of impairment associated with alcohol intoxication, are currently being employed by the Victoria Police (Australia) for the identification of driving impairment associated with drugs other than alcohol. The present thesis was designed to evaluate whether the SFSTs are a sensitive measure for identifying impairment associated with a single acute therapeutic amphetamine dose. Furthermore, the accuracy of using the SFSTs to detect driving impairment associated with these amphetamine doses was also evaluated. The present thesis examined the effects of a single acute therapeutic dose of various amphetamine preparations, on simulated driving performance, driving-related cognitive processes (assessed using standard cognitive tasks and the electroencephalogram [EEG]), and performance on the SFSTs, in healthy, stimulant-using, non-fatigued adults. The present thesis consisted of five separate experiments. The first three experiments examined the effects of d-amphetamine, d,l-methamphetamine, and d-methamphetamine, on simulated driving performance, driving-related cognitive processes, and performance on the SFSTs. Experiment 4 and Experiment 5 assessed the effects of d-amphetamine and dmethamphetamine on visual and auditory cognitive processes using the EEG. These forms of amphetamines were selected as they are commonly used recreationally by young adult drivers, and occupationally by truck drivers. Experiment 1, Experiment 2, and Experiment 3 employed a repeated-measures, counterbalanced, double blind, placebo-controlled design. In each experiment, twenty different (i.e. 60 participants in total) healthy volunteers (10 males and 10 females) completed two treatment conditions i) placebo and ii) 0.42mg/kg amphetamine (~30mg). Driving performance was assessed using a driving simulator task, which consisted of four driving tasks; 'freeway traffic driving' and 'city traffic driving' in both day and night conditions. Cognitive performance was assessed using a range of computer and pen and paper tasks designed to assess attention, psychomotor performance, and perceptual speed. Specifically, the tasks were: the Digit Span Test; a Digit Vigilance task; a Movement Estimation Task; the Digit Symbol Substitution Test; a Tracking Task; the Trail-Making Test; and the Inspection Time task. SFSTs performance was assessed using the Horizontal Gaze Nystagmus (HGN) test, the Walk and Turn (WAT) test, and the One Leg Stand (OLS) test. Three blood and saliva samples were obtained throughout all experimental sessions (120, 170, and 240 minutes after drug administration). The results indicated that 0.42mg/kg d-amphetamine significantly impaired simulated driving performance, in recreational stimulant users, 2-3 hours post-drug administration, when mean blood amphetamine concentrations were approximately 90ng/mL. No significant driving decrements were observed following d,l-methamphetamine or dmethamphetamine, when methamphetamine blood concentrations were 90ng/mL and 70ng/mL, respectively. There were only few driving behaviours that were found to be significantly reduced with d-amphetamine, such as reductions in signalling adherence and driving too fast for the traffic conditions. However, during all three amphetamine conditions, drivers travelled at a slower speed on the freeway at the time that an emergency situation occurred, relative to the placebo condition. It was argued that either this may result from more cautious driving, or that the reduction in speed acted as a compensatory mechanism to permit drivers to attend to other aspects of driving. Overall, the present results indicate that a therapeutic dose of amphetamine does not produce considerable impairment to driving, as only minimal amphetamine effects were observed on driving performance. In terms of cognitive performance, the results indicated that a therapeutic dose of various amphetamines has minimal effect on driving-related cognitive functioning, with some significant improvements noted in aspects of attention, psychomotor functioning and perceptual speed. This is consistent with the failure to identify significant driving impairments, described above, following a similar dose. However, the ability to perceive and predict motion and estimate 'time to contact', assessed using a movement estimation task, was affected following d-amphetamine and d-methamphetamine consumption. In terms of performance on the SFSTs, the present thesis demonstrated that following the administration of low-level d-amphetamine, d,l-methamphetamine, and dmethamphetamine, performance on the SFSTs was not impaired. Using the SFSTs, impairment associated with low dose d-amphetamine was identified in only 5% of cases, dmethamphetamine in 5% of cases, and d,l-methamphetamine in 0% of cases. These findings indicate that the degree of impairment produced with the low amphetamine dosing conditions was below the threshold of sensitivity of the SFSTs. However, as significant impairments in driving were not observed with amphetamines, the present SFSTs findings highlight that these tests are unlikely to produce false positive results during police drug evaluation procedures for amphetamine-related impairments. Experiment 4 and Experiment 5 similarly employed a repeated-measures, counterbalanced, double blind, placebo-controlled design. In each experiment, twenty healthy volunteers (10 males and 10 females) completed two treatment conditions i) placebo and ii) 0.42mg/kg amphetamine (~30mg). Tasks designed to assess visual and auditory cognitive functions relevant to driving were administered. Specifically, these processes were: divergent visual system pathways (magnocellular and parvocellular pathways); aspects of visual field processing (central and peripheral visual fields); mismatch negativity (MMN); prepulse inhibition (PPI); selective attention; resource allocation; and speed of processing. Two blood and saliva samples were obtained throughout all experimental session (120 and 200 minutes after drug administration). d-amphetamine and d-methamphetamine generally improved cognitive functioning, as assessed with visual and auditory ERP indices. Specifically, the results demonstrated that a low-level acute dose of d-amphetamine and d-methamphetamine improved early processing of visual information (indexed by improvements to the P100 component for the magnocellular and parvocellular visual pathways). In addition, d-methamphetamine improved the speed at which visual information was evaluated and processed (indexed by decreases in P300 latency), which was consistent with d-methamphetamine-related improvements in reaction time. There was a trend for d-amphetamine to improve the speed that changes in auditory stimulation were automatically detected (indexed by decreases in MMN latency). In addition, d-methamphetamine improved the ability to automatically 'screen out' irrelevant and intrusive auditory information (indexed by increases in PPI of the startle response). d-amphetamine was found to improve the speed at which auditory information was evaluated and processed (indexed by decreases in P300 latency), which was substantiated with corresponding improvements in reaction time and accuracy. Although amphetamines were generally shown to enhance ERP indices, a trend was found for d-amphetamine to differentially affect different regions of the visual field, in terms of selective attention. Specifically, there was a trend-level indication that d-amphetamine improved indices of selective attention (denoted by increases in N200 amplitude) for information presented centrally, but impaired indices of selective attention (denoted by decreases in N200 amplitude) for information presented in the periphery. Although impairments to the peripheral visual field were not similarly observed with dmethamphetamine, decrements to indices of selective attention (denoted by decreases in N200 amplitude) were also found with d-methamphetamine during the auditory oddball task. In terms of driving, these results suggest that drivers dosed with low-level amphetamine may not selectively attend to and discriminate changes within the traffic environment, although further research is required to confirm this. In conclusion, the present thesis has demonstrated that a single acute therapeutic dose of amphetamine produces minimal and inconsistent effects to driving. However, some (inconsistent) evidence was found that suggests that there may be mild impairments such as decreased ability to perceive and predict motion, tunnel vision effects, and decrements to selective attention. In addition, the present thesis highlights that at therapeutic doses, amphetamines do not impair SFSTs performance, which is in accordance with the failure to identify substantive amphetamine-related decrements to driving and cognitive functioning observed in the present thesis

Cooperativity of imprinted genes inactivated by acquired chromosome 20q deletions

Large regions of recurrent genomic loss are common in cancers; however, with a few well-characterized exceptions, how they contribute to tumor pathogenesis remains largely obscure. Here we identified primate-restricted imprinting of a gene cluster on chromosome 20 in the region commonly deleted in chronic myeloid malignancies. We showed that a single heterozygous 20q deletion consistently resulted in the complete loss of expression of the imprinted genes L3MBTL1 and SGK2, indicative of a pathogenetic role for loss of the active paternally inherited locus. Concomitant loss of both L3MBTL1 and SGK2 dysregulated erythropoiesis and megakaryopoiesis, 2 lineages commonly affected in chronic myeloid malignancies, with distinct consequences in each lineage. We demonstrated that L3MBTL1 and SGK2 collaborated in the transcriptional regulation of MYC by influencing different aspects of chromatin structure. L3MBTL1 is known to regulate nucleosomal compaction, and we here showed that SGK2 inactivated BRG1, a key ATP-dependent helicase within the SWI/SNF complex that regulates nucleosomal positioning. These results demonstrate a link between an imprinted gene cluster and malignancy, reveal a new pathogenetic mechanism associated with acquired regions of genomic loss, and underline the complex molecular and cellular consequences of “simple” cancer-associated chromosome deletions

E-SLAM HSCs do not expand in old JAK2^V617F knock-in mice and show reduced functional ability as well as a delayed entry into the cell cycle.

<p>(A) E-SLAM HSCs were increased in frequency in wild type (∼2-fold, see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001576#pbio-1001576-g001" target="_blank">Figure 1E</a>) but not JAK2^V617F marrow in 18–24-mo-old mice (<i>n</i> = 10) compared to 6–10-mo-old mice resulting in a 3-fold overall reduction in E-SLAM HSCs compared to wild-type (<i>p</i> = 0.002) in three independent experiments. (B) Individual HSCs were cultured and cell counts were recorded on day 1 and day 2 to determine whether or not they had undergone a division in three independent experiments. At day 2, significantly fewer (<i>p</i> = 0.039) old JAK2^V617F HSCs had divided. The cloning efficiency (C), number of cells per clone (D), and number of KSL cells per clone (F) were not different, but the JAK2^V617F cells still produced more differentiated cell types after 10 d of culture (<i>p</i> = 0.039, E). (G) Competitive transplantation of whole bone marrow from old JAK2^V617F mice, transformed JAK2^V617F mice, and their respective WT littermate controls. Relative chimerism is calculated by measuring donor chimerism as a percentage of donor+competitor chimerism and normalized to the average of the WT contribution (set to 1). The old JAK2^V617F BM displays reduced chimerism (<i>p</i><0.01), whereas transformed JAK2^V617F mice that have undergone transformation reacquire their self-renewal capacity.</p

JAK2^V617F HSCs have an initial survival advantage and make larger, more differentiated clones.

<p>(A) Schematic for single cell in vitro cultures. Individual CD45⁺/EPCR⁺/CD48⁻/CD150⁺ (E-SLAM) cells, obtained from mice 6–10 mo following pIpC injection, were sorted into single wells and cultured for 10 d in 300 ng/mL SCF and 20 ng/mL IL-11 in four independent experiments. (B) The average cloning efficiency was higher (<i>p</i> = 0.05) for JAK2^V617F (red bars) versus wild type (blue bars) cells and was measured by counting the number of sorted events that give rise to a colony after 10 d. (C) The average number of cells per clone was higher (<i>p</i> = 0.016) in JAK2^V617F cells. JAK2^V617F HSCs give rise to more differentiated cells (<i>p</i> = 0.006) as measured by the expression of one or more of a panel of lineage markers (CD5, Mac1, CD19, B220, Ly6g, 7-4, or Ter119, panel D) and expression of c-Kit and Sca1 as a surrogate for stem/progenitor cell number (E). Fourteen-day cultures of 100–400 E-SLAM HSCs in SCF+IL-11 followed by flow cytometric analysis of the cells show that, by proportion, JAK2^V617F HSCs make more CD41⁺ (<i>p</i> = 0.003, F), and less Ly6g/Mac1⁺ cells (<i>p</i> = 0.008, G) than wild-type controls in three independent experiments. The proportion of CD71⁺ cells generated was not changed (H). (I) The absolute numbers of Ly6g/Mac1⁺ and CD71⁺ cells generated were not different, but the number of CD41⁺ cells produced was increased approximately 2-fold (<i>p</i> = 0.023).</p

JAK2V617F induces a loss of self-renewal activity and HSC numbers and leads to a lineage bias when limited HSCs are transplanted.

<p>(A) Relative chimerism following transplantation of 10⁶ or 10⁵ JAK2^V617F or wild type (WT) whole bone marrow (BM) 6–10 months post-pIpC along with 5×10⁵ whole BM competitor cells into eight recipient mice. Average chimerism was lower in mice receiving JAK2^V617F cells (<i>p</i> = 0.03). (B) The relative myeloid (purple) versus lymphoid (green) contribution in each of the recipient animals were determined by calculating a ratio between the contribution to the myeloid compartment [Donor GM/(Donor GM+Competitor GM)] and lymphoid compartment [Donor BT/(Donor BT+Competitor BT)]. (C) Relative chimerism in primary peripheral blood (PB) (white bars) in the eight animals receiving 10⁵ cells compared to levels of PB chimerism in the 16 secondary recipients (two per primary animal, grey bars) in two independent transplantation experiments. An “X” represents a recipient that showed less than 1% chimerism at 24 wk posttransplantation, and a ∧ represents a recipient that only had contribution to the lymphoid lineages. (D) The relative myeloid (purple) versus lymphoid (green) contribution in each of the secondary recipient animals were determined by calculating a ratio between the contribution to the myeloid and lymphoid compartments as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001576#pbio-1001576-g001" target="_blank">Figure 1B</a>. (E) The FACS isolation strategy for CD45⁺/EPCR⁺/CD48⁻/CD150⁺ (E-SLAM) cells. The panels are gated on viable white blood cells and show E-SLAM gates for WT (top) and JAK2^V617F (bottom). (F) The frequency of E-SLAM HSCs per 10⁵ viable bone marrow (BM) cells in four WT and four JAK2^V617F mice 6–10 mo following pIpC injection from four independent experiments. The frequency is reduced in JAK2^V617F animals (<i>p</i> = 0.0288).</p

JAK2^V617F alters the balance of HSC fate choices.

<p>(A) A paired daughter cell analysis of WT and JAK2^V617F HSCs shows both daughters differentiate more often from JAK2^V617F parent HSCs than from WT HSCs as shown by measuring the percentage of KSL cells remaining after 10 d. Each paired daughter set is connected by a line and the pairs are categorized into symmetric SR (both daughters above the WT average %KSL), asymmetric division (one daughter above and one below the average %KSL), and symmetric differentiation (both daughters below the average %KSL). Note the relative increase in symmetric differentiation at the expense of asymmetric divisions. (B) The same paired daughter pairs are displayed here by the absolute number of KSL cells produced. Here it is clear that some of the JAK2^V617F pairs produce very few KSL cells (less than 100 per clone in some of the asymmetric divisions and symmetric differentiation divisions compared to WT HSCs, which are all above 100 KSL cells). (C) The pie graph on the left represents the outcome from 78 WT paired daughters (39 pairs), and the pie on the left represents the outcome from 76 mutant paired daughters (38 pairs). (D) Normally, HSCs will execute one of several programs in concert with the other HSCs to provide the requisite numbers of stem cells, progenitors, and differentiated cells for the organism. JAK2^V617F disturbs this balance and increases the likelihood of differentiation. As HSCs with the V617F mutation age, they have both an increased chance of fully exhausting as well as an increased chance of progressing to a more severe disease state, likely due to the acquisition of additional genetic or epigenetic perturbations.</p

Cultured JAK2^V617F E-SLAM HSCs produce more short-term progenitors, but lack long-term reconstitution ability.

<p>(A) Cells derived from cultures of 100–400 E-SLAM HSCs were harvested after 10 d of culture in SCF and IL-11 and then placed in a colony-forming cell (CFC) assay to determine the number and type of progenitor cells made or were transplanted into irradiated recipients to determine whether or not long-term reconstituting ability was retained. (B) Following 10–14 d of culture in the CFC assay, colonies were scored and enumerated. Colonies were scored as either Erythroid (E), Granulocyte/Macrophage (GM), or Granulocyte/Macrophage/Erythroid/Megakaryocyte (GEMM) progenitors and are represented by bar graphs showing the mean +/– SEM of four to six biological replicates from four independent experiments. A greater number of GM (<i>p</i> = 0.009) and E (<i>p</i> = 0.007) were observed in CFCs derived from JAK2^V617F cultures. (C) Varying doses (40, 33, 4) of HSC starting equivalents (the proportion of the total culture that would have been made by that input number of HSCs) were transplanted to determine the frequency of cells that had retained long-term reconstituting ability in two independent experiments. This is followed by a limiting dilution analysis that estimates the frequency of HSCs retained in the culture. Cultures of JAK2^V617F HSCs make 5–6-fold fewer HSCs in culture compared to WT littermate controls (<i>p</i> = 0.00469). * HSC dose is defined as the number of starting equivalents that were transplanted. In the case of “40,” this is representative of transplanting all of the cells that would be generated from a 10-d culture of 40 HSCs. ** A mouse was considered to be positive if it had >1% donor chimerism at 16–24 weeks and represented at least 0.5% of each lineage (GM, B, and T) at some point over the 16-wk period.</p