Abstract
Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (lin-, Sca-1+, c-Kit+) cells which closely resemble MPP1 cells. HPCLSK reconstitute hematopoiesis in lethally irradiated recipient mice over more than eight months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD or MLL-AF9 their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSK can be applied to transgenic mice and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, Cdk6-/- HPCLSKs induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells.
Key points
We describe the generation of murine cell lines (HPCLSK) which reliably mimic hematopoietic/leukemic progenitor cells.
Cdk6-/- BCR/ABLp210 HPCLSKs uncover a novel role for CDK6 in homing.
INTRODUCTION
Adult hematopoietic stem cells (HSCs) represent 0.01-0.005% of all nucleated cells in the bone marrow (BM). They are unique in their ability to continuously self-renew, differentiate into distinct lineages of mature blood cells1 and regenerate a functional hematopoietic system following transplantation into immunocompromised mice2–5. Most hematopoietic malignancies originate in stem/progenitor cells upon acquirement of genetic/epigenetic defects. These so called leukemic stem cells (LSCs) maintain key characteristics of regular HSCs, including the ability of self-renewing and multi-potency6,7.
Although hematopoietic cell differentiation is a dynamic and continuous process, cell surface marker expression defining distinct subsets and developmental stages is an inevitable tool in HSC characterization. A common strategy is to further define murine lineage negative, c-Kit and Sca-1 positive (LSK) cells by their CD48, CD135, CD150 and CD34 expression. This marker combination stratifies the most dormant HSCs into the increasingly cycling multipotent progenitors (MPP) 1 and 2 and the myeloid or lymphoid prone MPP3 and 48. Leukemia, analogous to normal hematopoiesis, is hierarchically organized; LSCs residing in the BM initiate and maintain the disease and give rise to their more differentiated malignant progeny. Therapeutically, LSCs are often resistant against many current cancer treatments and thus cause disease relapse9–13. Understanding potential Achilles’ heels in LSCs to develop new curative therapeutic approaches is of fundamental interest and represents a major frontier of cancer biology.
Understanding hematopoietic disease development and defining therapeutic intervention sites requires the availability of multi-potential hematopoietic cell lines. HSCs can be maintained and expanded to a very limited extent in vitro - the vast majority of their progeny differentiates in culture. Numerous attempts have been made to increase the number of long-term (LT)-HSCs in culture including the use of high levels of cytokines and growth factors or ill-defined factors secreted by feeder cells14–28.
Alternatively, immortalization using genetic manipulation was employed to establish stem cell-like cell lines. One major limitation of these cell lines is the failure to reconstitute a fully functional hematopoietic system upon transplantation29–30. One of the most successful immortalized murine multipotent hematopoietic cell lines is the EML (Erythroid, Myeloid, and Lymphocytic) line derived by retroviral expression of a truncated, dominant-negative form of the human retinoic acid receptor. However, EML cells are phenotypically and functionally heterogeneous and display a block in the differentiation of myeloid cells31–38.
An alternative route for immortalization of murine multipotent hematopoietic cells was employing Lhx239–41, a LIM-homeobox domain transcription factor binding a variety of transcriptional co-factors. Lhx2 is expressed in embryonic hematopoietic locations such as the aorta-gonad-mesonephros (AGM) region, yolk sac and fetal liver, but is absent in BM, spleen and thymus of adult mice42–44. Lhx2 up-regulates key transcriptional regulators for HSCs including Hox and Gata while down-regulating differentiation-associated genes39. Lhx2 is aberrantly expressed in human chronic myelogenous leukemia suggesting a role for Lhx2 in the growth of immature hematopoietic cells45. Enforced expression of Lhx2 in BM-derived murine HSCs and embryonic stem cells (ES)/induced pluripotent (iPS) cells resulted in ex vivo expansion of engraftable HSC-like cells41,42,46 strictly dependent on stem cell factor (SCF) and yet undefined autocrine loops providing additional secreted molecule(s)40. These cells generate functional progeny and long term repopulate stem cell–deficient hosts40,43,47–48. The cyclin-dependent kinase 6 (CDK6) has been recently described as a critical regulator of HSC quiescence and is essential in BCR/ABLp210 LSCs49–50. Besides its main characteristic, CDK6 and its close homolog CDK4 control cell cycle progression, CDK6 functions as a transcriptional regulator51–53. CDK6 is recognized as being a key oncogenic driver in hematopoietic malignancies and therefore represents a promising target for cancer therapy and intervention49,54–56. More recent evidence highlights the importance of CDK6 during stress, including oncogenic transformation when CDK6 counteracts p53 effects57. Furthermore, CDK6 plays a crucial role in several myeloid diseases, including Jak2V617F+ MPN, CML and AML by regulating stem cell quiescence, apoptosis, differentiation and cytokine secretion49,56,58–59.
Using the long term culture system, it was possible to generate HPCLSKs from the transgenic mouse line Cdk6-/- which represents a powerful tool to analyze specific functions of CDK6 in progenitor cells and allows mechanistic and therapeutic studies tailored specifically to leukemic stem/progenitors cells.
MATERIALS AND METHODS
Animals
Mice (C57BL/6N, NSG [NOD.Cg-Prkdcscid Il2rgtm 1Wjl/SzJ], Ly5.1+ [B6.SJL-Ptprca]) and Cdk6-/-60 were bred and maintained under special pathogen-free (SPF) conditions at the Institute of Pharmacology and Toxicology, University of Veterinary Medicine, Vienna, Austria. Age-matched (7-11 weeks) male and female mice were used unless indicated otherwise. All procedures were approved by the institutional ethics and animal welfare committee (BMWFW-68.205/0093-WF/V/3b/2015 and BMWFW-68.205/0112-WF/V/3b/2016) and the national authority according to §§26ff. of the Animal Experiment Act, Tierversuchsgesetz 2012 - TVG 2012.
HPCLSK cell line generation
BM of two to five C57BL/6 mice was isolated, pooled and sorted for LSK cells. Sorted LSK cells were cultured in 48-well-plates for 48 hours in a 1:1 ratio of Stem Pro-34 SFM (Gibco/ Thermo Scientific, Waltham, MA, USA) and Iscoves modified Dulbecco medium (IMDM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.75□×□10−4 M 1-Thiolglycerol (MTG, Sigma), Penicillin/Streptomycin (P/S, Sigma), 2 mM L-Glutamine (L-Glut, Sigma), 25 U heparin (Sigma), 10 ng fibroblast growth factor (mFGF) acidic (R&D Sytems, Minneapolis, USA), 10 ng mIGF-II (R&D), 20 ng mTPO (R&D), 10 ng mIL-3 (R&D), 20 ng hIL-6 (R&D) and stem fell factor (SCF, generated in-house) used at 2% final concentration. LSK cells were transduced with a Lhx2 pMSCV-puromycin (Clontech/Takara, Mountain View, CA, USA) vector47 in 1% pegGOLD Universal Agarose (Peqlab/VWR Darmstadt, Germany) coated 48-well-plates and transfected four times on day three to six with the Lhx2- containing viral supernatant. At day seven, cells were transferred to 1% agarose-coated 24-well-plates in IMDM with 5% FCS, 1.5□×□10-4 M MTG, P/S, 2 mM L-Glut referred hereafter as IMDM culture medium. Additionally, the IMDM culture medium was supplemented with 12.5 ng/ml IL-6 (R&D) and 2% SCF. At day ten, 1.5 μg/ml puromycin (InvivoGen, San Diego, USA) was added to the medium to select for the Lhx2 expressing LSK cells. The same reagents were subsequently used for all the experiments.
HPCLSK cell line culture
HPCLSK cell lines were kept on 1% agarose coated culture plates. Solidified plates were stored in a 5% CO2 humidified incubator with 1 ml IMDM culture media per well. HPCLSK cells were plated in IMDM culture media supplemented with 12.5 ng/ml IL-6, 2% SCF and 1.5 μg/ml puromycin on the agarose plates. Cells were continuously kept at a density between 0.8-2□×□106 cells/ml. BM-derived HPC5 cell line was kept in IMDM culture media supplemented with 12.5 ng/ml IL-6 and 2% SCF, while BM-derived HPC9 cells and ES cell line-derived HPC-7 were cultured in IMDM supplemented with 2% SCF (all lines provided by Leif Carlsson). The virus packaging cell lines Platinum-E (Plat-E,Cell Biolabs, Inc, San Diego, CA, USA) and GPp210-GFP61 were kept in DMEM (Sigma) supplemented with 10% FCS and P/S. The pre-pro-B-BCR/ABLp185-GFP were cultured in RPMI (Sigma) with 10% FCS and P/S57.
RESULTS
Generation of murine hematopoietic progenitor HPCLSK cell lines
To meet the increasing need of studying hematopoietic stem/progenitor cells, we sought to establish a robust method to generate murine stem-cell lines by modifying a strategy that was originally described by the Carlsson lab41,47. Sorted murine LSK cells were maintained in cytokine- and growth factor-supplemented serum-free medium for 2 days. Thereafter, the cells were infected with a retroviral construct encoding Lhx2 coupled to a puromycin selection marker and switched to SCF, IL-6 and 5% serum-containing IMDM medium on agarose-coated plates to prevent attachment-induced differentiation. Puromycin selection was initiated ten days after sorting. Within four weeks continuously proliferating, HPCLSK cell lines establish and can be stored long term by cryopreservation (Fig. 1a). LSK cells can be classified into dormant HSCs and four subsequent MPP populations based on their surface markers8. HPCLSK cell lines express c-Kit and Sca-1 but lack expression of the myeloid and lymphoid lineage markers Gr-1 (neutrophil), CD11b (monocyte/macrophage), CD3 (T cell), CD19 (B cell) and Ter119 (erythroid). According to the CD34, CD48 and CD150 expression, HPCLSKs categorize as MPP2 – a population able to give rise to myeloid and lymphoid cells8. Despite the MPP2 surface expression markers, transcriptome analysis of HPCLSKs revealed a predominant overlap with the MPP1 signature pointing to an even more immature state. Upon long-term culture a uniform cellular morphology is maintained within the cell lines (Fig 1b, Supplementary Fig. S1a-d). Comparison to other progenitor cell lines including the bone marrow derived BM-HPC5, BM-HPC9 and the ES-derived HPC-7 cell line41 showed that HPCLSKs have the most immature profile. The other cell lines are either positive for lineage markers or lack Sca-1 expression. The ES-derived HPC-7 cell line stains positive for c-Kit, Sca-1, CD48 and CD150 and lacks lineage markers. It is also limited in its differentiation capacity62–63 (Supplementary Fig. 1e).
HPCLSK cells are able to differentiate to myeloid and lymphoid cells in vitro
To explore signaling patterns, HPCLSK cells were treated with cytokines for 15 min. EPO, GM-CSF or IL-3 resulted in phosphorylation and activation of STAT5, STAT3, AKT and ERK signaling, while IL-6 induced predominantly STAT3 phosphorylation. STAT3, AKT and ERK were also activated upon SCF treatment albeit to a lesser extent in line with signaling in stem/progenitor cells (Fig. 1c). In line, HPCLSK cells formed erythroid (BFU-E), myeloid (CFU-GM, CFU-GEMM) and pre-B (CFU-preB) cell colonies in methylcellulose enriched cytokines (EPO, GMCSF, IL-7, SCF, IL-6, IL-3) comparable to primary BM-derived cells (Fig. 1d-e). We confirmed expression of erythroid (Ter119/CD71), myeloid (CD11b/Gr-1) or B cell (B220/CD93) markers on these colonies (Supplementary Fig 1g). In comparison, the ability to form colonies and to in vitro differentiate of HPC-7 and BM-HPC5 cells was reduced in accordance with an impaired cytokine–induced activation of STAT5, STAT3, AKT and ERK (Supplementary Fig. S1h-j).
HPCLSKs are multipotent in vivo
As HPCLSKs differentiate into myeloid and lymphoid lineages in vitro, we explored the potential of the cells to protect mice from radiation-induced death in vivo. Lethally irradiated Ly5.1+ mice received 1×107 Ly5.2+ BM-HPC5 or HPCLSK cells per tail vein injection. Ly5.2+ BM cells were used as controls. Non-injected irradiated mice died within 10 days, briefly thereafter followed by BM-HPC5 recipients. Injection of HPCLSKs and injection of primary BM cells rescued the mice due to the efficient repopulation of the hematopoietic system (Fig. 2a-b). After 40 days, white blood cell (WBC) and red blood cell (RBC) counts were comparable between HPCLSKs–injected and BM-injected controls (Fig. 2c). Blood counts remained stable over a 6-months-period after which the experiment was terminated (Supplementary Fig. S2a). HPCLSKs had efficiently homed to the BM, blood, spleen and thymus comparable to the BM control and no alterations of the spleen weight was detectable (Fig. 2d-e). FACS analysis confirmed the efficient repopulation of the hematopoietic system. Numbers of myeloid and lymphoid progenitors in the BM and differentiated blood cells (Gr-1+ granulocytes, CD11b+ monocytes, Gr-1/CD11b+ eosinophils/neutrophils and B220+ B cells) were comparable to BM-injected mice. Only HPCLSK-derived CD4+ or CD8+ T cells were significantly lower in the blood, however, were present in the thymus in similar numbers as in the BM-injected control (Fig. 2f).
To determine cell numbers required for hematopoietic repopulation in mice, we gradually lowered the cell number used for injection. 2.5×106 HPCLSKs sufficed to allow for an 80% survival of the animals for a period of at least 8 months, after which the experiment was terminated. Injection of 1×106 HPCLSKs did not induce long-term survival but significantly prolonged the lifespan of lethally irradiated animals (median survival: 51 days compared to 8.5 days) (Supplementary Fig. S2b and S2c). These experiments led us to conclude that HPCLSKs possess the ability for long-term replenishment of the hematopoietic system.
Generation of leukemic HPCLSKs as a model for leukemic stem cells (LSCs)
LSCs differ from the bulk of leukemic cells and possess the ability for self-renewal. To establish LSC models, we infected HPCLSKs with a retrovirus encoding for oncogenes either inducing myeloid (BCR/ABLp210, MLL–AF9, Flt3-ITD;NRasG12D) or lymphoid (BCR/ABLp185) leukemia (Fig. 3a). Analysis of signaling pathways in the GFP+ leukemic lines showed that the cells faithfully reflected the signaling patterns downstream of the respective oncogene. As described, BCR/ABL predominantly induced phosphorylation of CRKL and STAT561,64. Flt3-ITD;NRasG12D was associated with a pronounced JAK2, STAT5, AKT and ERK signaling activation65 and MLL-AF9 upregulated c-MYC66 (Fig. 3b). In the presence of SCF and IL-6, transformed HPCLSKs retained the expression of stem cell markers. A small fraction of the cells differentiated and upregulated the respective lineage markers. BCR/ABL positive LSCs were able to grow cytokine independently, whereas other oncogenes are shown with SCF (Fig. 3c-d). Except for MLL-AF9, all oncogenes tested formed growth factor-independent colonies in methylcellulose gel (Supplementary Fig. S3a).
To determine their leukemic potential in vivo, transformed HPCLSKs were injected intravenously (i.v.) into NSG mice (Fig. 4a, left). HPCLSKs BCR/ABLp185 inflicted disease within 12 days, followed by HPCLSKs BCR/ABLp210 and HPCLSKs Flt3-ITD;NRasG12D which succumbed to disease within 50 days. The longest disease latency was observed upon injection of HPCLSKs MLL-AF9 which induced disease after three months (Fig. 4a, right). All diseased animals displayed elevated WBC counts, blast-like cells in the blood and suffered from splenomegaly (Fig. 4b, 4d, Supplementary Fig. S4a). GFP+ transformed HPCLSK cells were detected in the blood, spleen and BM of the diseased mice (Fig. 4c). HPCLSKs BCR/ABLp210, HPCLSKs MLL-AF9 and HPCLSKs FLT3/NRasG12D-injected animals suffered from myeloid leukemia with an average of 92% CD11b+ cells, whereas HPCLSKs BCR/ABLp185-injected NSGs developed predominantly GFP+ B cells with a percentage mean of 32% of CD19+ cells (Fig. 4e, Supplementary Fig. S4b-d). These experiments determine HPCLSK cells as a valid model system studying leukemogenesis in vivo downstream of several oncogenic drivers.
HPCLSKs from a transgenic mouse strain – Cdk6-/- HPCLSKs
CDK6 plays a key role as a transcriptional regulator for HSC activation and its function extends to LSCs49. To gain insights into distinct functions of CDK6 in HSCs/LSCs, we generated HPCLSK cell lines from Cdk6-/- transgenic mice60. CDK4 does not compensate for the loss of CDK6 in those lines (Supplementary Fig. 5a). Cdk6-/- HPCLSKs grow under normal HPCLSK culture conditions albeit with a reduced cell proliferation and slightly increased apoptosis when compared to wild type HPCLSKs (Fig. 5a, Supplementary Fig. 5b). 5×106 Cdk6+/+ or Cdk6-/- HPCLSKs were equally well capable to rescue lethally irradiated mice for up to 60 days (data not shown).
In a murine CML model BCR/ABLp210 Cdk6-/- BM cells induced disease significantly slower and with a drastically reduced disease phenotype49. To investigate whether this phenotype can be recapitulated with HPCLSKs, we generated Cdk6+/+ and Cdk6-/- BCR/ABLp210 HPCLSKs by retroviral infection. Irrespective of the presence of CDK6, BCR/ABLp210 HPCLSKs grow in the absence of any cytokine and retain the expression of LSK markers (Supplementary Fig. 5c). In line with murine CML models, Cdk6-/- BCR/ABLp210 HPCLSKs form fewer growth-factor independent colonies when compared to Cdk6+/+ controls 7 days after plating, yet the difference did not reach significance (Fig. 5b)49. BCR/ABLp210 HPCLSK-derived colonies displayed Gr-1 and CD11b marker expression. However, Cdk6-/- BCR-ABLp210 HPCLSKs show a trend to higher Gr-1 and lower CD11b expression compared to wild type (Supplementary Fig. S5d). To study the leukemic potential of Cdk6-/- BCR/ABLp210 HPCLSKs in vivo, we injected 1*106 cells i.v. into NSG mice. Cdk6+/+ BCR/ABLp210 HPCLSKs inflict disease within 14 days with severe signs of leukemia, including splenomegaly (Fig. 5c, Supplementary Fig. S5e). In contrast, Cdk6-/- BCR/ABLp210 HPCLSKs failed to induce disease within this time period and only two thirds of the mice started to show signs of disease around 80 days after injection whereas one third of the animals did not develop any sign of leukemia within 7 months. Analysis of diseased mice show a reduced infiltration of Cdk6-/- BCR/ABLp210 HPCLSKs into the BM and spleen, the percentage of BCR/ABLp210 GFP+ cells is comparable to Cdk6+/+ control cells (Fig. 5d, Supplementary Fig. S5f). These results underline the crucial role of CDK6 in BCR/ABLp210 LSCs and verify the potential of our novel cellular HPCLSK system to charter leukemic phenotypes.
CDK6 dependent transcript alterations
To study CDK6-dependent gene regulation in untransformed and BCR/ABLp210 transformed HPCLSKs, we performed RNA-Seq analysis. Untransformed HPCLSKs lacking CDK6 show an altered gene regulation with 1335 genes up- and 661 genes down-regulated when compared to Cdk6+/+ HPCLSKs (Fig. 6a). These differences decreased upon transformation; cytokine-independent BCR/ABLp210 HPCLSKs showed 85 up- and 468 genes down-regulated in the absence of CDK6 compared to controls. Overall, 80% and 40% of genes found to be up- or downregulated in Cdk6-/- BCR/ABLp210 HPCLSK cells were also de-regulated in Cdk6-/- untransformed HPCLSK cells defining a transformation-independent gene signature downstream of CDK6 (Fig. 6B). Gene Ontology enrichment analyses of CDK6 dependent genes revealed an association with immune response, cell adhesion, cell death and myeloid cell differentiation irrespective of the transformation status (Fig. 6C). The differential gene expression in our murine BCR/ABLp210 HPCLSK cells was compared to CDK6 associated gene expression changes in human CML samples. To do so, we stratified a dataset from 76 human CML patients into CDK6high and CDK6low samples based on quartile expression of CDK6 and subsequently calculated the differential gene expression. We identified 101 genes that are regulated in a CDK6–dependent manner in murine and human BCR/ABLp210 cells (Fig. 6D). In human and mouse CDK6 dependent deregulated genes belong to pathways pointing at apoptosis/stress response, cell differentiation and homing.
Validation of CDK6 dependent pathways in LSCs
In line with the deregulated pathways in human and mouse resulting from the RNA-Seq analysis, we recently demonstrated that CDK6 regulates apoptosis during BCR/ABLp185 transformation57. To validate this aspect in our HPCLSK system, we serum starved Cdk6+/+ and Cdk6-/- BCR/ABLp210 HPCLSKs for 90 minutes and performed an apoptosis staining by flow cytometry (Fig. 7a). As expected, Cdk6-/- BCR/ABLp210 HPCLSKs showed increased response to stress.
In addition to apoptosis, cell differentiation was one of the most significant deregulated pathways detected by the transcriptome analysis. Colonies from Cdk6-/- BCR/ABLp210 HPCLSKs showed a bias to the granulocytic direction by increased Gr-1 expression (Supplementary Fig. S5c). In the RNA-Seq analysis and validated by qPCR, Csf3r, an essential receptor for granulocytic differentiation, is upregulated in Cdk6 -/- BCR/ABLp210 cells compared to controls (Fig. 7b). Further, cytokine independent Cdk6-/- BCR/ABLp210 HPCLSKs show increased mean fluorescence intensity (MFI) levels of Gr1 and reduced MFI levels of CD11b compared to Cdk6+/+ controls (Fig. 7C). Together, these data demonstrate that loss of CDK6 shows an advantage for granulocytic differentiation.
Last but not least, the reduced percentages of Cdk6-/- BCR/ABLp210 HPCLSKs in the BM and spleen upon i.v. injection (Fig. 5d) together with the RNA-Seq analysis point towards a hampered homing capacity of Cdk6-/- BCR/ABLp210 HPCLSK cells. We validated several deregulated genes found in the transcriptome analysis which can be linked to homing by qPCR analysis (Fig. 7D) and performed an in vivo homing assay. To do so, we injected 1*106 BCR/ABLp210 HPCLSKs with and without CDK6 into aged and gender matched female C57BL/6N mice and profiled the number of BCR/ABLp210 GFP+ cells after 18h in the BM and spleen by flow cytometry. Cdk6-/- BCR/ABLp210 HPCLSKs showed a significantly diminished homing capability to the BM compared to Cdk6+/+ BCR/ABLp210 HPCLSKs.
Taken together, the validated data describes essential roles of CDK6 in LSCs and supports the strong reliability of our murine cellular system. Moreover, we here describe a prominent function for CDK6 in regulating BCR/ABLp210 leukemic cell homing.
DISCUSSION
Functional and molecular studies on hematopoietic and leukemic stem cells have provided numerous insights into the mechanisms of hematopoietic diseases. However, progress is restricted by the limited availability of hematopoietic stem/progenitor cells and the difficulty of in vitro culturing. We present a robust procedure to generate an unlimited source of functional mouse HSC/HPC lines called HPCLSK that possess characteristics of MPPs and can serve as a source of lymphoid and myeloid LSC lines. HPCLSKs are multipotent cells that retain lymphoid and myeloid differentiation potential and can repopulate lethally irradiated mice without supporter BM cells. More than 90% of HPCLSKs are Lin-/c-Kit+/Sca-1+ and express CD34, CD48 and CD150, which is characteristic of MPP2. They also express CD41, which marks cells at the embryonic AGM that constitute the myelo-erythroid and myelo-lymphoid branchpoint in early hematopoiesis67–68. The transcriptome of the cells most closely resembles that of MPP1 cells, which correspond to the earliest proliferating stem/progenitor cell.
Our approach is robust and simple and requires no co-culture system or feeder layer and no extensive amounts of cytokines. We have established more than 50 distinct HPCLSK cell lines with an efficiency of 100%, using either mouse strains of various genetic backgrounds or transgenic mice as a source. HPCLSK cells can be genetically modified by retroviral transduction or CrispR/Cas9 technologies, so are a versatile tool in HSC and LSC research. Our method is based on the enforced expression of Lhx2, a transcription factor for mouse HPC immortalization39,41–42,46–47. Improvements to the original protocol include FACS sorting of LSKs to avoid 5-FU treatment, the use of serum low-media with a defined cocktail of cytokines, pre-coating of plates to avoid adherence–induced myeloid differentiation and the maintenance of high HPCLSK cell density4,41,47,69–75. Lhx2-immortalized HPCs have been reported to induce a transplantable myeloproliferative disorder resembling human chronic myeloid leukemia in long-term engrafted mice76. We did not observe this even after long-term repopulation in lethally irradiated Ly5.1 or in immunosuppressed NSG mice. The difference probably stems from our use of sorted LSKs instead of total BM to overexpress Lhx2, as the myeloid disorder may originate from a more differentiated myeloid progenitor. We have used HPCLSKs as a source to generate leukemic stem cells and obtained leukemic HPCLSK lines harboring BCR/ABL, MLL-AF9 and Flt3-ITD;NRasG12D oncogenes. Removal of SCF and IL-6 in vitro induced myeloid differentiation, indicating that the self-renewal program depends on the presence of low-level cytokines and downstream signaling events that are provided in vivo by the BM niche.
The cell cycle kinase CDK6 is a transcriptional regulator and is particularly important in hematopoietic malignancies. In HSCs, its actions are largely independent of its kinase activity. It is essential for HSC activation in the most dormant stem cell population under stress situations, including transplantation and oncogenic stress. The impact of CDK6 extends to leukemic stem cells, as BCR/ABLp210-transformed BM cells fail to induce disease in vivo in the absence of CDK6. To investigate how CDK6 drives leukemogenesis in progenitor cells, we generated Cdk6-/- HPCLSKs from Cdk6-deficient mice and transformed them with BCR/ABLp210. The absence of CDK6 was associated with a reduced incidence of leukemia and with significantly delayed disease development, thereby mimicking the effects seen in primary bone marrow transplantation assays. RNA-Seq and subsequent pathway analysis show deregulated stress response, cell adhesion and apoptotic processes/cell death in the absence of CDK6. This result is consistent with our recent observations that CDK6 antagonizes p53 responses and regulates survival. In the absence of CDK6, hematopoietic cells need to overcome oncogenic-induced stress by mutating p53 or activating alternative survival pathways, as in the case of Cdk6-deficient JAK2V617F positive LSKs. Another featured shared by CDK6-deficient JAK2V617F+ LSKs and CDK6-deficient BCR/ABL HPCLSK is an altered cytokine secretion, as revealed by pathway enrichment analysis in both systems. HSCs show homing and cell adhesion, which allow them to migrate to the bone marrow and replenish hematopoietic lineages77. GO pathway analysis revealed deregulated cell adhesion and cell migration pathways in HPCLSK cell lines and in human patient samples. Our bioinformatic data show that loss of CDK6 from transformed cells leads to a significantly reduced capacity to home to the bone marrow, which slows the onset of leukemic disease. The common CDK6 dependent gene signature between BCR/ABLp210 HPCLSKs and human CML patient samples underlines the translational relevance of our model system. A large subset of CDK6 regulated genes is also found in patients, which we could validate with specific assays using our BCR/ABLp210 HPCLSKs. The data strengthen our confidence in our murine cellular system and show that results from HPCLSK experiments can be translated to the human situation. HPCLSK lines thus represent a quick and simple alternative to the lymphoid progenitor Ba/F3 or the myeloblast-like 32D cells to explore the potential transforming ability of mutations found in hematopoietic malignancies.
Author contributions
ED and IMM designed and conducted experiments, collected and analyzed data. TB, BM and IM collected and analyzed data. RG, MZ and GH performed bio-informatical analysis. LC was involved in conception and design of the study, contributed essential material and reviewed the manuscript. KK designed and supervised experiments. AHK reviewed the manuscript and supervised experiments. VS designed and supervised the study, VS, ED, IMM, BM and KK wrote the manuscript.
Funding
This work was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme grant agreement No 694354. This work was supported by the Austrian Science Foundation (FWF) via grants to K.K. (P 31773).
Conflict of interest
The authors declare that they have no conflicts of interest.
SUPPLEMENTARY FIGURE LEGENDS
Acknowledgements
We thank P. Kudweis, S. Fajmann, M. Ensfelder-Koparek and P. Jodl for excellent technical support and M. Dolezal for critical discussion of bioinformatical analysis. We thank the Biomedical Sequencing Facility (BSF) at CeMM for NGS library preparation, sequencing and related bioinformatics analyses.