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

Single-cell approaches identify the molecular network driving malignant hematopoietic stem cell self-renewal.

Recent advances in single-cell technologies have permitted the investigation of heterogeneous cell populations at previously unattainable resolution. Here we apply such approaches to resolve the molecular mechanisms driving disease in mouse hematopoietic stem cells (HSCs), using JAK2V617F mutant myeloproliferative neoplasms (MPNs) as a model. Single-cell gene expression and functional assays identified a subset of JAK2V617F mutant HSCs that display defective self-renewal. This defect is rescued at the single HSC level by crossing JAK2V617F mice with mice lacking TET2, the most commonly comutated gene in patients with MPN. Single-cell gene expression profiling of JAK2V617F-mutant HSCs revealed a loss of specific regulator genes, some of which were restored to normal levels in single TET2/JAK2 mutant HSCs. Of these, Bmi1 and, to a lesser extent, Pbx1 and Meis1 overexpression in JAK2-mutant HSCs could drive a disease phenotype and retain durable stem cell self-renewal in functional assays. Together, these single-cell approaches refine the molecules involved in clonal expansion of MPNs and have broad implications for deconstructing the molecular network of normal and malignant stem cells.MS is the recipient of a BBSRC Industrial CASE PhD Studentship, and CAO and JF are recipients of Wellcome Trust PhD Studentships. Work in the Kent lab is supported by a Bloodwise Bennett Fellowship (15008), a European Hematology Association Non-Clinical Advanced Research Fellowship, and an ERC Starting Grant (ERC-2016-STG–715371). Work in the Green Lab is supported by the Wellcome Trust, Bloodwise, Cancer Research UK, the Kay Kendall Leukaemia Fund, and the Leukemia and Lymphoma Society of America. Dr. Kent, Professor Göttgens, and Professor Green are all supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute the National Institute for Health Research Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre

Mutant calreticulin knockin mice develop thrombocytosis and myelofibrosis without a stem cell self-renewal advantage.

Somatic mutations in the endoplasmic reticulum chaperone calreticulin (CALR) are detected in approximately 40% of patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF). Multiple different mutations have been reported, but all result in a +1-bp frameshift and generate a novel protein C terminus. In this study, we generated a conditional mouse knockin model of the most common CALR mutation, a 52-bp deletion. The mutant novel human C-terminal sequence is integrated into the otherwise intact mouse CALR gene and results in mutant CALR expression under the control of the endogenous mouse locus. CALRdel/+ mice develop a transplantable ET-like disease with marked thrombocytosis, which is associated with increased and morphologically abnormal megakaryocytes and increased numbers of phenotypically defined hematopoietic stem cells (HSCs). Homozygous CALRdel/del mice developed extreme thrombocytosis accompanied by features of MF, including leukocytosis, reduced hematocrit, splenomegaly, and increased bone marrow reticulin. CALRdel/+ HSCs were more proliferative in vitro, but neither CALRdel/+ nor CALRdel/del displayed a competitive transplantation advantage in primary or secondary recipient mice. These results demonstrate the consequences of heterozygous and homozygous CALR mutations and provide a powerful model for dissecting the pathogenesis of CALR-mutant ET and PMF

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.This work was supported by the Leukemia and Lymphoma Society grant, the UK Medical Research Council (Roger Pedersen), the National Institute for Health Research (NIHR; RP-PG-0310-1002; Willem Ouwehand and Cedric Ghevaert) and a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute. Cedric Ghevaert is supported by the British Heart Foundation (FS/09/039); Marloes Tijssen is supported by the European Hematology Association (Research fellowship) and the British Heart Foundation (PG/13/77/30375). Catherine Hobbs was supported by the National Health Service Blood and Transplant. Matthew Trotter was supported by a Medical Research Council Centre grant (MRC Centre for Stem Cell Biology and Regenerative Medicine); since participation in the work described, Matthew Trotter has become an employee of Celgene Research SLU, part of Celgene Corporation. Nicole Soranzo's research and Sanger Institute affiliates are supported by the Wellcome Trust (WT098051 and WT091310), the EU FP7 (Epigenesys 257082 and Blueprint HEAL TH-F5-2011-282510). The Cambridge Biomedical Centre (BRC) hIPSCs core facility is funded by the NIHR.This is the final version of the article. It first appeared from Nature Publishing Group via https://doi.org/10.1038/ncomms1120

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases like Huntington’s disease. Autophagy induction with the mTOR inhibitor, rapamycin, accelerates clearance of these toxic substrates. As rapamycin has non-trivial side effects, we screened FDA-approved drugs to identify novel autophagy-inducing pathways. We found that L-type Ca(2+) channel antagonists, the K(+)(ATP) channel opener minoxidil, and the G(i) signaling activator clonidine, induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, where cAMP regulates IP(3) levels, influencing calpain activity, which completes the cycle by cleaving and activating G(sα), which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced and we provide proof-of-principle for therapeutic relevance in Huntington’s disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca(2+), like excitotoxicity, will inhibit autophagy, thus retarding clearance of aggregate-prone proteins

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

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

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