JAK2V617F Mutant Megakaryocytes Contribute to Hematopoietic Aging in a Murine Model of Myeloproliferative Neoplasm

Introduction Recent studies implicated megakaryocytes (MKs) in regulating hematopoietic stem cell (HSC) function. Abnormal MK hyperplasia is a hallmark feature of myeloproliferative neoplasms (MPNs). In the present study, we investigated the effects of JAK2V617F-bearing MKs on HSC aging in a murine model of MPN during a 2-yr follow up. Methods JAK2V617F Flip-Flop (FF1) mice (which carry a Cre-inducible human JAK2V617F gene driven by the human JAK2 promoter) and Pf4-Cre mice (which express Cre under the promoter of platelet factor 4, a MK-specific gene) were crossed to generate MK lineage-specific human JAK2V617F transgenic mouse line (Pf4 +FF1 +). Results During aging, the Pf4 +FF1 +mice maintained an essential thrombocythemia phenotype with no evidence of transformation to leukemia or myelofibrosis (Figure 1). Myeloid-biased HSCs (Lin -cKit +Sca1 +CD150 +CD48 -CD41 +) were significantly expanded in 1yr old and 2yr old Pf4 +FF1 +mice compared to age-matched control mice, while no difference in myeloid-biased HSC numbers was detected between young (6mo) Pf4 +FF1 +mice and control mice. Competitive repopulation assays and serial transplantation assays showed that HSCs from old Pf4 +FF1 +mice had a reduced engraftment and self-renewal capacity with a skewed differentiation towards the myeloid lineage. Results from the serial transplantation experiments also indicated that the functional decline of HSCs in aged Pf4 +FF1 +mice were HSC-intrinsic and was not reversible. Both cell cycle analysis by Hoechst33342 and Pyronin Y staining and cell proliferation analysis by in vivo BrdU labeling revealed that the JAK2V617F mutant MK niche can promote hematopoietic aging in old Pf4 +FF1 +mice by increasing HSC proliferation/cycling. Taken together, the Pf4 +FF1 +mice demonstrated several hallmarks of accelerated HSC aging.(Figure 2) Next, we examined the hematopoietic microenvironment in the old Pf4 +FF1 + mice. Using whole-mount immunofluorescent staining and confocal imaging, we found that the cKit +HSPCs were located further from MKs in old Pf4 +FF1 +mice compared to old control mice. We also found that CD45 -CD31 +Sca1 - sinusoidal marrow endothelial cell (EC) number (by flow cytometry analysis) and marrow vascular area (by in vivo VE-cadherin staining) were significantly decreased in aged Pf4 +FF1 +mice compared to control mice. Tube formation assays confirmed that conditioned medium from old Pf4 +FF1 +MK culture significantly inhibited EC angiogenesis in vitro. These findings suggest that the JAK2V617F mutant MK niche not only altered its own interaction with HSCs during aging, but also suppressed the vascular niche function to promote HSC aging. (Figure 3) To further understand the mechanisms by which JAK2V617F mutant MKs promote HSC aging, we performed both RNA sequencing and targeted cytokine arrays of wild-type and JAK2V617F mutant MKs from both young (6mo) and old (2yr) Pf4-cre control and Pf4 +FF1 +mice. We found that HSC aging had a profound effect on MK transcriptomic profiles and dysregulated pathways in cell adhesion molecules, MAPK signaling, NF-kappa B signaling, hematopoietic cell lineage, and cytokine-cytokine receptor interaction were highly upregulated in old JAK2V617F mutant MKs compared to young JAK2V617F mutant MKs. FAS ligand, IL-12, tissue inhibitor of metalloproteinases-1, IL-10, IL-6, MIG, macrophage inflammatory protein 1a, GCSF, IL-5, MIP-1g were produced in increased amounts in old mutant MKs compared to old wild-type MKs. Many of these factors are involved in inflammation, angiogenesis, extracellular matrix remodeling, and hematopoiesis regulation. Sensitive PCR assays confirmed that human JAK2 gene was expressed in MKs but not in HSCs in 2yr old Pf4 +FF1 +mice. In vitro co-culture and in vivo co-transplantation assays provided further evidence that the JAK2V617F mutant MKs affected wild-type HSC function directly. Therefore, the hematopoietic aging phenotype we have observed was not caused by any direct effect of the JAK2V617F mutation on HSC function because the Pf4 promoter was 'leaky'. Conclusions Results from this study support that, as a hematopoietic niche cell, MKs represent an important connection between the extrinsic and intrinsic mechanisms for HSC aging in MPNs -- the JAK2V617F-bearing MKs can alter the hematopoietic niche to accelerate HSC aging, and HSC aging in turn can profoundly remodel the niche e.g. by affecting MK transcriptomics. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

SRSF2-P95Hdelays Myelofibrosis Development through Altered JAK/STAT Signaling in JAK2-V617F Megakaryocytes

Background: The gain-of-function JAK2 V617F mutant is the most common driver mutation identified in myeloproliferative neoplasms (MPNs). Additional somatic variants, also found in other malignant hemopathies, are detected in primary myelofibrosis (MF) and supposed to contribute to fibrosis or leukemia development. One of these mutations affects SRSF2, a gene encoding a component of the splicing machinery. SRSF2 heterozygous mutation mainly affects the proline 95 residue of the protein. Its association with JAK2 V617F correlates with a reduced leukemia free survival. Whether and how SRSF2 P95 variants could favor fibrosis development in JAK2 V617F cells remained unknown. Methods & Results: To investigate how homozygous Jak2 V617F and heterozygous Srsf2 P95H could interact in the hematopoietic tissue, we generated conditional knock-in mice in which the CreERt recombinase expression was driven by the HSC-Scl promoter leading to Jak2 V617F and/or Srsf2 P95H hematopoietic-specific expression upon tamoxifen induction. Srsf2 P95H mutation initially exhibited limited effect on Jak2 V617F-induced polycythemia vera (PV) only slightly reducing erythrocytosis and leukocytosis (through a previously described decrease in B220 + B cell number). The expansion of hematopoietic stem cells (SLAM), multipotent progenitors (MPP) and megakaryocyte progenitors (MKP) observed in Jak2 V617F mice was not affected by Srsf2 status. However, while platelet count was decreasing in Jak2 V617F alone mice at later time point due to fibrosis development, Srsf2 P95H/Jak2 V617F combination further increased platelet counts correlating with a significant delay in the development of myelofibrosis. Bone marrow cells (BM) were transplanted into lethally irradiated recipient mice together with GFP-positive wild-type competitor cells and tamoxifen was administrated after transplantation. Double mutant cells initially demonstrated a limited competitive advantage over wild-type cells as compared to Jak2 V617F-only cells. However, serial transplantation revealed a rapid exhaustion of Jak2 V617F single mutant cells leading to lethal pancytopenia, which was not observed in animals transplanted with Jak2 V617F/Srsf2 P95H double mutant cells. As both monocytes and megakaryocytes (MK) were involved in fibrosis development, we further explored the role of these two cell populations. Spectral flow analysis of monocyte subsets in peripheral blood and BM failed to detect any significant change in double compared to single mutant animals. In contrast, double-mutant mice presented a significant delay in MK maturation with normalized expression of c-Mpl and ploidy. Using mass cytometry, we found ex vivo a higher proportion of MKP and MK expressing high levels of P-Stat5 in Jak2 V617F mice, which the addition of Srsf2 P95H tend to reduce, suggesting an altered Mpl/Jak2 signaling pathway. To validate the hypothesis that Srsf2 P95H negatively interfere with Jak2-mediated signaling in MK, we injected high dose of the thrombopoietin-mimetic romiplostim in mice transplanted with wild-type or Srsf2 P95H BM. Both thrombocytosis and myelofibrosis were significantly reduced in Srsf2 P95H transplanted animals. To further decipher the mechanism by which Srsf2 P95H could alter cell signaling, we performed bulk RNA sequencing on sorted MK. Pathway analysis using gene set enrichment analysis identified mostly a down-regulation of signaling pathways, including JAK/STAT signaling, in Jak2 V617F/Srsf2 P95H compared to Jak2 V617F single mutant cells. Further analysis of splicing events in Srsf2 P95H mutant cells identified an increased exon 14 skipping in Jak2, which was validated by RT-qPCR. Summary: Contrary to EZH2 mutation that promotes JAK2 V617F-induced myelofibrosis in mouse models, heterozygous Srsf2 P95H delays myelofibrosis development in Jak2 V617F-transgenic mice. Srsf2 P95H co-mutation prevents the clonal exhaustion induced by serial transplantation of JAK2 V617F BM cells. This effect is associated with a reduced signaling in MK, which may involve abnormal splicing of signaling components including Jak2 exon 14 skipping. Disclosures Abdel-Wahab: H3B Biomedicine: Consultancy, Research Funding; Foundation Medicine Inc: Consultancy; Merck: Consultancy; Prelude Therapeutics: Consultancy; LOXO Oncology: Consultancy, Research Funding; Lilly: Consultancy; AIChemy: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Envisagenics Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Loss of MBD2 attenuates MLL-AF9-driven leukemogenesis by suppressing the leukemic cell cycle via CDKN1C

Acute myeloid leukemia (AML) is a deadly cancer characterized by an expanded self-renewal capacity that is associated with the accumulation of immature myeloid cells. Emerging evidence shows that methyl-CpG-binding domain protein 2 (MBD2), a DNA methylation reader, often participates in the transcriptional silencing of hypermethylated genes in cancer cells. Nevertheless, the role of MBD2 in AML remains unclear. Herein, by using an MLL-AF9 murine model and a human AML cell line, we observed that loss of MBD2 could delay the initiation and progression of leukemia. MBD2 depletion significantly reduced the leukemia burden by decreasing the proportion of leukemic stem cells (LSCs) and inhibiting leukemia cell proliferation in serial transplantation experiments, thereby allowing leukemic blasts to transition to a more mature state reflecting normal myelopoiesis. Both gene expression analyses and bioinformatic studies revealed that MBD2 negatively modulated genes related to myeloid differentiation, and was necessary to sustain the MLL-AF9 oncogene-induced gene program. We further demonstrated that MBD2 could promote LSC cell cycle progression through epigenetic regulation of CDKN1C transcription probably by binding to its promoter region. Taken together, our data suggest that MBD2 promotes AML development and could be a therapeutic target for myeloid malignancies.

Correction: Pardo-Sánchez et al. Increased Tumor Growth Rate and Mesenchymal Properties of NSCLC-Patient-Derived Xenograft Models during Serial Transplantation. Cancers 2021, 13, 2980

The authors would like to make a correction to their published paper [...]

Engraftment characterization of risk-stratified AML patients in NSGS mice

Acute myeloid leukemia (AML) is the commonest acute leukemia in adults. Disease heterogeneity is well-documented and patient stratification determines treatment decisions. Patient-derived xenografts (PDXs) of risk-stratified AMLs are crucial for studying AML biology and testing novel therapeutics. Despite recent advances in PDX modeling of AML, reproducible engraftment of human AML is mainly limited to high-risk (HR) cases, with inconsistent or very protracted engraftment observed for favorable-risk (FR) and intermediate-risk (IR) patients. We have characterized the engraftment robustness/kinetics in NSGS mice of 28 AML patients grouped according to molecular/cytogenetic classification, and have assessed whether the orthotopic co-administration of patient-matched bone marrow mesenchymal stromal cells (BM-MSCs) improves AML engraftment. PDX event-free survival correlated well with the predictable prognosis of risk-stratified AML patients. The majority (85%-94%) of the mice were engrafted in BM independently of the risk group, although HR-AML patients showed engraftment levels significantly superior to those of FR- and IR-AML patients. Importantly, the engraftment levels observed in NSGS mice by week 6 remained stable overtime. Serial transplantation and long-term culture-initiating cell (LTC-IC) assays revealed long-term engraftment limited to HR-AML patients, fitter leukemia-initiating cells (LICs) in HR- than in FR- or IR-AML samples, and the presence of AML-LICs in the CD34- leukemic fraction, regardless the risk group. Finally, orthotopic co-administration of patient-matched BM-MSCs with AML cells resulted dispensable for BM engraftment levels but favored peripheralization of engrafted AML cells. This comprehensive characterization of human AML engraftment in NSGS mice offers a valuable platform for in vivo testing of targeted therapies in risk-stratified AML patient samples.

Increased Tumor Growth Rate and Mesenchymal Properties of NSCLC-Patient-Derived Xenograft Models during Serial Transplantation

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. The high mortality is very often a consequence of its late diagnosis when the cancer is already locally advanced or has disseminated. Advances in the study of NSCLC tumors have been achieved by using in vivo models, such as patient-derived xenografts. Apart from drug screening, this approach may also be useful for study of the biology of the tumors. In the present study, surgically resected primary lung cancer samples (n = 33) were implanted in immunodeficient mice, and nine were engrafted successfully, including seven adenocarcinomas, one squamous-cell carcinoma, and one large-cell carcinoma. ADC tumors bearing the KRAS-G12C mutation were the most frequently engrafted in our PDX collection. Protein expression of vimentin, ezrin, and Ki67 were evaluated in NSCLC primary tumors and during serial transplantation by immunohistochemistry, using H-score. Our data indicated a more suitable environment for solid adenocarcinoma, compared to other lung tumor subtypes, to grow and preserve its architecture in mice, and a correlation between higher vimentin and ezrin expression in solid adenocarcinomas. A correlation between high vimentin expression and lung adenocarcinoma tumors bearing KRAS-G12C mutation was also observed. In addition, tumor evolution towards more proliferative and mesenchymal phenotypes was already observed in early PDX tumor passages. These PDX models provide a valuable platform for biomarker discovery and drug screening against tumor growth and EMT for lung cancer translational research.

Cancer of unknown primary stem-like cells model multi-organ metastasis and unveil liability to MEK inhibition

Cancers of unknown primary (CUPs), featuring metastatic dissemination in the absence of a primary tumor, are a biological enigma and a fatal disease. We propose that CUPs are a distinct, yet unrecognized, pathological entity originating from stem-like cells endowed with peculiar and shared properties. These cells can be isolated in vitro (agnospheres) and propagated in vivo by serial transplantation, displaying high tumorigenicity. After subcutaneous engraftment, agnospheres recapitulate the CUP phenotype, by spontaneously and quickly disseminating, and forming widespread established metastases. Regardless of different genetic backgrounds, agnospheres invariably display cell-autonomous proliferation and self-renewal, mostly relying on unrestrained activation of the MAP kinase/MYC axis, which confers sensitivity to MEK inhibitors in vitro and in vivo. Such sensitivity is associated with a transcriptomic signature predicting that more than 70% of CUP patients could be eligible to MEK inhibition. These data shed light on CUP biology and unveil an opportunity for therapeutic intervention.

Using the Microwell-mesh to culture microtissues in vitro and as a carrier to implant microtissues in vivo into mice

Prostate cancer (PCa) patient-derived xenografts (PDXs) are commonly propagated by serial transplantation of “pieces” of tumour in mice, but the cellular composition of pieces is not standardised. Herein, we optimised a microwell platform, the Microwell-mesh, to aggregate precise numbers of cells into arrays of microtissues, and then implanted the Microwell-mesh into NOD-scid IL2γ−/− (NSG) mice to study microtissue growth. First, mesh pore size was optimised using microtissues assembled from bone marrow-derived stromal cells, with mesh opening dimensions of 100×100 μm achieving superior microtissue vascularisation relative to mesh with 36×36 μm mesh openings. The optimised Microwell-mesh was used to assemble and implant PCa cell microtissue arrays (hereafter microtissues formed from cancer cells are referred to as microtumours) into mice. PCa cells were enriched from three different PDX lines, LuCaP35, LuCaP141, and BM18. 3D microtumours showed greater in vitro viability than 2D cultures, but neither proliferated. Microtumours were successfully established in mice 81% (57 of 70), 67% (4 of 6), 76% (19 of 25) for LuCaP35, LuCaP141, and BM18 PCa cells, respectively. Microtumour growth was tracked using live animal imaging for size or bioluminescence signal. If augmented with further imaging advances and cell bar coding, this microtumour model could enable greater resolution of PCa PDX drug response, and lead to the more efficient use of animals. The concept of microtissue assembly in the Microwell-mesh, and implantation in vivo may also have utility in implantation of islets, hair follicles or other organ-specific cells that self-assemble into 3D structures, providing an important bridge between in vitro assembly of mini-organs and in vivo implantation.

Serial transplantation unmasks galectin-9 contribution to tumor immune escape in the MB49 murine model

Mechanisms of tumor immune escape are quite diverse and require specific approaches for their exploration in syngeneic tumor models. In several human malignancies, galectin-9 (gal-9) is suspected to contribute to the immune escape. However, in contrast with what has been done for the infiltrating cells, the contribution of gal-9 produced by malignant cells has never been demonstrated in an animal model. Therefore, we derived isogenic clones—either positive or negative for gal-9—from the MB49 murine bladder carcinoma cell line. A progressive and consistent reduction of tumor growth was observed when gal-9-KO cells were subjected to serial transplantations into syngeneic mice. In contrast, tumor growth was unaffected during parallel serial transplantations into nude mice, thus linking tumor inhibition to the enhancement of the immune response against gal-9-KO tumors. This stronger immune response was at least in part explained by changing patterns of response to interferon-γ. One consistent change was a more abundant production of CXCL10, a major inflammatory factor whose production is often induced by interferon-γ. Overall, these observations demonstrate for the first time that serial transplantation into syngeneic mice can be a valuable experimental approach for the exploration of novel mechanisms of tumor immune escape.

After migration into blood circulation, hematopoietic stem cells can stably self-renew and maintain bone marrow while being skewed toward myeloid lineage when submitted to serial transplantation

Hematopoietic stem cells (HSCs) mainly reside in bone marrow (BM) within niches providing an appropriate environment for their survival and self-renewal. Although, small numbers of HSCs can quit their residing environment to migrate into blood circulation and re-engraft elsewhere in BM. Mobilizing agents such as granulocyte colony stimulating factor (G-CSF) can amplify this process by inducing massive HSC mobilization into blood circulation. This method is widely used in clinics to treat hematological disorders. However, in physiological conditions, the properties of HSCs after migration (called migratory HSCs) remain incompletely characterized. In this study, we investigated the capacity of migratory HSCs to self-renew, reconstitute and maintain BM. We show that after migration, HSCs can stably self-renew and maintain BM in homeostasis. However, while stably repopulating BM of irradiated recipients, migratory HSCs show a defect in lymphoid lineage reconstitution when subjected to serial transplantations. Our findings provide interesting knowledge on HSC properties after migration, which may benefits therapeutic research on HSC-based therapies to treat hematological disorders.