Lineage Transdifferentiation Research Articles

Use of a dual genetic system to decipher exocrine cell fate conversions in the adult pancreas

Unraveling cell fate plasticity during tissue homeostasis and repair can reveal actionable insights for stem cell biology and regenerative medicine. In the pancreas, it remains controversial whether lineage transdifferentiation among the exocrine cells occur under pathophysiological conditions. Here, to address this question, we used a dual recombinase-mediated genetic system that enables simultaneous tracing of pancreatic acinar and ductal cells using two distinct genetic reporters, avoiding the “ectopic” labeling by Cre-loxP recombination system. We found that acinar-to-ductal transdifferentiation occurs after pancreatic duct ligation or during caerulein-induced pancreatitis, but not during homeostasis or after partial pancreatectomy. On the other hand, pancreatic ductal cells contribute to new acinar cells after significant acinar cell loss. By genetic tracing of cell proliferation, we also quantify the cell proliferation dynamics and deduce the turnover rate of pancreatic exocrine lineages during homeostasis. Together, these results suggest that the lineage transdifferentiation happens between acinar cells and ductal cells in the pancreatic exocrine glands under specific conditions.

Therapy-Induced Transdifferentiation Promotes Glioma Growth Independent of EGFR Signaling.

EGFR is frequently amplified, mutated, and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical responses, and the underlying mechanism remains poorly understood. Using an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals that a small population of glioma cells can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses identified TGFβ/YAP/Slug signaling cascade activation as a major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, TGFβ secreted from stressed glioma cells acted to promote YAP nuclear translocation that stimulated upregulation of the pro-mesenchymal transcriptional factor SLUG and subsequent glioma lineage transdifferentiation toward a stable therapy-refractory state. Blockade of this adaptive response through suppression of TGFβ-mediated YAP activation significantly delayed anti-EGFR relapse and prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses. SIGNIFICANCE: This study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and are clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas with aberrant EGFR signaling.

CBMS-09 Intercellular communication at glioblastoma stem cell niches

Glioblastoma multiforme (GBM) contains heterogenous population of cells including a small population of GBM stem cells (GSCs), which potentially cause therapeutic resistance and tumor recurrence. GSCs harbored in special microenvironments, such as perinecrotic niche, perivascular niche, border niche. However, the mechanisms underlying the pathogenesis and maintenance of GSCs remain largely unknown. Stemness and chemo-radioresistance was promoted by not only additional mutation, but also microenvironment of GBM cells. Previously, we had reported that growth factors and cytokines secreted by oligodendrocyte lineage cells and macrophages/microglia induce stemness and chemo-radioresistance into GBM cells. Recently, Ito et al. reported that incorporation of ribosomes and ribosomal proteins into somatic cells promoted lineage trans-differentiation toward multipotency. Ribosomal proteins exist intra- and extracellularly. There is a possibility that ribosomal proteins promote stemness into cancer cells, we focused on 40S ribosomal protein S6 (RPS6), which is related to cell proliferation in lung and pancreatic cancer, but not reported in GBM. RPS6 was significantly upregulated in high-grade glioma. SiRNA-mediated RPS6 knock-down significantly suppressed the characteristics of GSCs, including their tumorsphere potential and stemness marker expression, such as Nestin and Sox2. RPS6 overexpression enhanced the tumorsphere potential of GSCs. Moreover, RPS6 expression was significantly correlated with SOX2 expression in different glioma grades. Immunohistochemistry data indicated that RPS6 was predominant detected at GSC niches, concurrently with the data from IVY GAP databases. Furthermore, RPS6 and other ribosomal proteins were upregulated in GSC-predominant areas in this database. The present results indicate that, in GSC niches, ribosomal proteins play crucial roles in the development and maintenance of GSCs and are clinically associated with chemo-radioresistance and GBM recurrence. These results suggested that intercellular communications through growth factors, cytokines, and ribosomes are regarded as new treatment targets of GBM.

Ribosomal protein S6 promotes stem-like characters in glioma cells.

Glioblastoma multiforme (GBM), a lethal brain tumor developing in the white matter of the adult brain, contains a small population of GBM stem cells (GSCs), which potentially cause chemotherapeutic resistance and tumor recurrence. However, the mechanisms underlying the pathogenesis and maintenance of GSCs remain largely unknown. A recent study reported that incorporation of ribosomes and ribosomal proteins into somatic cells promoted lineage trans‐differentiation toward multipotency. This study aimed to investigate the mechanism underlying stemness acquisition in GBM cells by focusing on 40S ribosomal protein S6 (RPS6). RPS6 was significantly upregulated in high‐grade glioma and localized at perivascular, perinecrotic, and border niches in GBM tissues. siRNA‐mediated RPS6 knock‐down significantly suppressed the characteristics of GSCs, including their tumorsphere potential and GSC marker expression; STAT3 was downregulated in GBM cells. RPS6 overexpression enhanced the tumorsphere potential of GSCs and these effects were attenuated by STAT3 inhibitor (AG490). Moreover, RPS6 expression was significantly correlated with SOX2 expression in different glioma grades. Immunohistochemistry data herein indicated that RPS6 was predominant in GSC niches, concurrent with the data from IVY GAP databases. Furthermore, RPS6 and other ribosomal proteins were upregulated in GSC‐predominant areas in this database. The present results indicate that, in GSC niches, ribosomal proteins play crucial roles in the development and maintenance of GSCs and are clinically associated with chemoradioresistance and GBM recurrence.

Abstract 5191: Functional role of EZH2 in neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is the most aggressive malignant variant of prostate cancer (PCa). Since there is no effective treatment available for NEPC to date, devising a therapeutic intervention requires identifying the mechanisms of how prostate cancer cells gain neuroendocrine phenotype. Evidence suggest that neuroendocrine differentiation is associated with elevated expression of Enhancer of Zeste Homolog 2 (EZH2). EZH2 is the catalytic subunit of Polycomb repressive complex 2 (PRC2) that functions to regulate histone H3 methylation and causes transcriptional repression of target genes. We hypothesize that the elevated expression of EZH2 in NEPC may render the tumors cells an undifferentiated state which enables the lineage switch and trans-differentiation into neuroendocrine malignancies. Method and Results: First, we extracted microarray data to analyze the expression of polycomb genes during PCa progression, using Gene Expression Omnibus (GEO) database from NCBI. The expressions of Polycomb genes (EZH2, EED, SUZ2, CBX1) were up-regulated in metastatic castrate-resistant prostate cancer and the increased EZH2 expression was in concert with elevated expressions of NEPC marker (CHGA, FOXA2, SOX2) and decreased expression of FOXA1, a prostate differentiation marker. Additionally, we conducted IHC staining in sections derived from benign prostate hyperplasia, low- and high- grade prostate adenocarcinoma, patient-derived NEPC xenograft tumors (LuCaP), and mouse models of NEPC (12T10 LADY and TRAMP). EZH2 and FOXA2 were overexpressed in all NEPC tumors while FOXA1 was abundant in prostate adenocarcinoma but decreased in NEPC. Also, we cultured prostate adenocarcinoma, LNCaP cells in androgen-depleted media and analyzed the gene expression of major polycomb genes and NEPC markers by qPCR. The expressions of Polycomb genes (BMI1, RING1a, CBX1) were induced by androgen depletion, concurrent with the induction of NEPC gene, CD56. Furthermore, we stably knocked down EZH2 in DU145 and PC3 cell lines (both have NEPC features) using shRNA and measured both the RNA and protein level of EZH2, PCa and NEPC markers using qPCR and western blot. We found that the levels of AR, FOXA1 and E-cadherin are upregulated in both DU145 and PC3 cells when EZH2 was knocked down. Conclusion: Our data support that EZH2 is functionally involved in the development of NEPC. Future direction: We will overexpress EZH2 in LNCaP cells to study if elevated EZH2 expression promotes NEPC. We will also perform RNA-seq and ChIP-seq to study how the altered expression of EZH2 affects the global transcriptome and identify the downstream target genes of EZH2 in PCa. Additionally, we will conduct in vivo animal experiments to determine EZH2’s role in NEPC using NEPC PDX models and transgenic mouse models. Findings from this research would provide detailed insights into developing therapeutics against EZH2 mediated NEPC. (Funding source: R03 and FWCC). Citation Format: Md Imtiaz Khalil, Shu Yang, Anthony Blankenship, Zachary Connelly, Xiuping Yu. Functional role of EZH2 in neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5191.

SUMO Safeguards Somatic and Pluripotent Cell Identities by Enforcing Distinct Chromatin States

Understanding general principles that safeguard cellular identity should reveal critical insights into common mechanisms underlying specification of varied cell types. Here, we show that SUMO modification acts to stabilize cell fate in a variety of contexts. Hyposumoylation enhances pluripotency reprogramming invitro and invivo, increases lineage transdifferentiation, and facilitates leukemic cell differentiation. Suppressing sumoylation in embryonic stem cells (ESCs) promotes their conversion into 2-cell-embryo-like (2C-like) cells. During reprogramming to pluripotency, SUMO functions on fibroblastic enhancers to retain somatic transcription factors together with Oct4, Sox2, and Klf4, thus impeding somatic enhancer inactivation. In contrast, in ESCs, SUMO functions on heterochromatin to silence the2C program, maintaining both proper H3K9me3 levels genome-wide and repression of the Dux locus by triggering recruitment of the sumoylated PRC1.6 and Kap/Setdb1 repressive complexes. Together, these studies show that SUMO acts on chromatin as a glue to stabilize key determinants of somatic and pluripotent states.

Myeloid transformation of plasma cell myeloma: molecular evidence of clonal evolution revealed by next generation sequencing

BackgroundPlasma cell myeloma (PCM) is a neoplasm of terminally differentiated B lymphocytes with molecular heterogeneity. Although therapy-related myeloid neoplasms are common in plasma cell myeloma patients after chemotherapy, transdifferentiation of plasma cell myeloma into myeloid neoplasms has not been reported in literature. Here we report a very rare case of myeloid neoplasm transformed from plasma cell myeloma.Case presentationA 60-year-old man with a history of plasma cell myeloma with IGH-MAF gene rearrangement and RAS/RAF mutations developed multiple soft tissue lesions one year following melphalan-based chemotherapy and autologous stem cell transplant. Morphological and immunohistochemical characterization of the extramedullary disease demonstrated that the tumor cells were derived from the monocyte-macrophage lineage. Next generation sequencing (NGS) studies detected similar clonal aberrations in the diagnostic plasma cell population and post-therapy neoplastic cells, including IGH-MAF rearrangement, multiple genetic mutations in RAS signaling pathway proteins, and loss of tumor suppressor genes. Molecular genetic analysis also revealed unique genomic alterations in the transformed tumor cells, including gain of NF1 and loss of TRAF3.ConclusionTo our knowledge, this is the first case of myeloid sarcoma transdifferentiated from plasma cell neoplasm. Our findings in this unique case suggest clonal evolution of plasma cell myeloma to myeloma neoplasm and the potential roles of abnormal RAS/RAF signaling pathway in lineage switch or transdifferentiation.

Ribosome Incorporation into Somatic Cells Promotes Lineage Transdifferentiation towards Multipotency

Recently, we reported that bacterial incorporation induces cellular transdifferentiation of human fibroblasts. However, the bacterium-intrinsic cellular- transdifferentiation factor remained unknown. Here, we found that cellular transdifferentiation is caused by ribosomes. Ribosomes, isolated from both prokaryotic and eukaryotic cells, induce the formation of embryoid body-like cell clusters. Numerous ribosomes are incorporated into both the cytoplasm and nucleus through trypsin-activated endocytosis, which leads to cell-cluster formation. Although ribosome-induced cell clusters (RICs) express several stemness markers and differentiate into derivatives of all three germ layers in heterogeneous cell populations, RICs fail to proliferate, alter the methylation states of pluripotent genes, or contribute to teratoma or chimera formation. However, RICs express markers of epithelial–mesenchymal transition without altering the cell cycle, despite their proliferation obstruction. These findings demonstrate that incorporation of ribosomes into host cells induces cell transdifferentiation and alters cellular plasticity.

Preexisting endothelial cells mediate cardiac neovascularization after injury.

The mechanisms that promote the generation of new coronary vasculature during cardiac homeostasis and after injury remain a fundamental and clinically important area of study in the cardiovascular field. Recently, it was reported that mesenchymal-to-endothelial transition (MEndoT) contributes to substantial numbers of coronary endothelial cells after myocardial infarction. Therefore, the MEndoT has been proposed as a paradigm mediating neovascularization and is considered a promising therapeutic target in cardiac regeneration. Here, we show that preexisting endothelial cells mainly beget new coronary vessels in the adult mouse heart, with essentially no contribution from other cell sources through cell-lineage transdifferentiation. Genetic-lineage tracing revealed that cardiac fibroblasts expand substantially after injury, but do not contribute to the formation of new coronary blood vessels, indicating no contribution of MEndoT to neovascularization. Moreover, genetic-lineage tracing with a pulse-chase labeling strategy also showed that essentially all new coronary vessels in the injured heart are derived from preexisting endothelial cells, but not from other cell lineages. These data indicate that therapeutic strategies for inducing neovascularization should not be based on targeting presumptive lineage transdifferentiation such as MEndoT. Instead, preexisting endothelial cells appear more likely to be the therapeutic target for promoting neovascularization and driving heart regeneration after injury.

Differentiation of mesenchymal stem cells derived from human bone marrow and subcutaneous adipose tissue into pancreatic islet-like clusters in vitro

Although stem cells are present in various adult tissues and body fluids, bone marrow has been the most popular source of stem cells for treatment of a wide range of diseases. Recent results for stem cells from adipose tissue have put it in a position to compete for being the leading therapeutic source. The major advantage of these stem cells over their counterparts is their amazing proliferative and differentiation potency. However, their pancreatic lineage transdifferentiation competence was not compared to that for bone marrow-derived stem cells. This study aims to identify an efficient source for transdifferentiation into pancreatic islet-like clusters, which would increase potential application in curative diabetic therapy. The results reveal that mesenchymal stem cells (MSC) derived from bone marrow and subcutaneous adipose tissue can differentiate into pancreatic islet-like clusters, as evidenced by their islet-like morphology, positive dithizone staining and expression of genes such as Nestin, PDX1, Isl 1, Ngn 3, Pax 4 and Insulin. The pancreatic lineage differentiation was further corroborated by positive results in the glucose challenge assay. However, the results indicate that bone marrow-derived MSCs are superior to those from subcutaneous adipose tissue in terms of differentiation into pancreatic islet-like clusters. In conclusion, bone marrow-derived MSC might serve as a better alternative in the treatment of diabetes mellitus than those from adipose tissue.

Beating in a dish: new hopes for cardiomyocyte regeneration

Functional human cardiomyocytes hold great promise in cell transplantation-based therapy to treat many heart diseases. To meet this devastating and clinical need, researchers are infatuated with developing novel technologies and methodologies to efficiently generate cardiomyocytes through either stem cell differentiation or cell lineage transdifferentiation. Though exciting progress has been made, challenges remain to be addressed before the translation from bench side to bed side can be fulfilled.

Langerhans cell sarcoma with an aberrant cytoplasmic CD3 expression

Langerhans cell sarcoma is a rare and aggressive high grade hematopoietic neoplasm with a dismal prognosis. It has a unique morphological and immunotypic profile with a CD1a/ langerin/S100 + phenotype. T cell lineage markers except for CD4 in Langerhans cell sarcoma have not been documented previously. We report a case of 86 year-old male of Caucasian descent who presented with an enlarging right neck mass over 2 months with an underlying unknown cause of anemia. Computed tomography scan of the neck, chest and abdomen revealed generalized lymphadenopathy and mild splenomegaly suspicious for lymphoma. Diagnostic core biopsy performed on right neck mass revealed a possible T cell lymphoma with expression of T cell lineage specific marker CD3 but conclusive diagnosis could not be made due to insufficient core biopsy sample. Further excisional biopsy performed on a left inguinal node showed a hematopoietic neoplasm with features of Langerhans cell sarcoma with a focal cytoplasmic CD3 expression in 30-40% of the tumor cells. PCR for T cell receptor (TCR) gene rearrangement failed to demonstrate a clonal gene rearrangement in the tumor cells arguing against a T cell lineage transdifferentiation, suggesting an aberrant CD3 expression. To the best of our knowledge, this case represents the first report of Langerhans cell sarcoma with an aberrant cytoplasmic CD3 expression.Virtual slideshttp://www.diagnosticpathology.diagnomx.eu/vs/2065486371761991

Modulation of PKC‐α promotes lineage reprogramming of committed B lymphocytes

During hematopoietic lineage development, hematopoietic stem cells sequentially commit toward myeloid or lymphoid lineages in a tightly regulated manner, which under normal circumstances is irreversible. However, studies have established that targeted deletion of the B-lineage specific transcription factor, paired box gene 5 (Pax5), enables B cells to differentiate toward other hematopoietic lineages, in addition to generating progenitor B-cell lymphomas. Our previous studies showed that subversion of protein kinase C (PKC)-α in developing B cells transformed B-lineage cells. Here, we demonstrate that PKC-α modulation in committed CD19(+) B lymphocytes also promoted lineage conversion toward myeloid, NK-, and T-cell lineages upon Notch ligation. This occurred via a reduction in Pax5 expression resulting from a downregulation of E47, a product of the E2A gene. T-cell lineage commitment was indicated by the expression of T-cell associated genes Ptcra, Cd3e, and gene rearrangement at the Tcrb gene locus. Importantly, the lineage-converted T cells carried Igh gene rearrangements reminiscent of their B-cell origin. Our findings suggest that modulation of PKC-α induces hematopoietic-lineage plasticity in committed B-lineage cells by perturbing expression of critical B-lineage transcription factors, and deregulation of PKC-α activity/expression represents a potential mechanism for lineage trans-differentiation during malignancies.

Histiocytic sarcoma transdifferentiated from follicular lymphoma presenting as a cutaneous tumor

Histiocytic sarcoma represents a rare and poorly understood tumor of histiocytic/dendritic cell lineage that can rarely present in the skin. Previously reported cases of histiocytic sarcoma after follicular lymphoma suggested that follicular lymphoma can transdifferentiate into histiocytic sarcoma. We describe another case involving a 40-year old male who developed histiocytic sarcoma in his right thigh 4 years after the diagnosis of grade 1 follicular lymphoma in the left neck. The two neoplasms were morphologically and immunophenotypically different but had identical immunoglobulin heavy chain gene and bcl-2 gene rearrangements, as demonstrated by polymerase chain gene reaction analysis, and the presence of t(14;18)(q32;q21) translocation was confirmed via fluorescence in situ hybridization (FISH) analysis. Because of spindle cell morphology and focal S-100 positivity, malignant peripheral nerve sheath tumor and melanoma diagnoses were made initially and extensive workup was required to discover the correct diagnosis. Lineage transdifferentiation can occur in mature lymphoid neoplasms and awareness of this phenomenon and appropriate workup is crucial for correct diagnosis, as different treatment protocols and prognosis may vary.

Reprogramming of Mouse Fibroblasts to an Intermediate State of Differentiation by Chemical Induction

Induced pluripotent stem cells (iPSCs) generated by forced expression of four transcription factors offer promises for regenerative and therapeutic uses in human diseases. However, it is necessary to overcome the risk of tumorigenicity caused by the use of potent oncogenes and the use of randomly integrating vectors before the iPSC technology can be applied to human medicine. Stem cells and cancer cells share many features in common, implying that there are similar underlying mechanisms in their development. Small molecules have been used to induce cell reprogramming for lineage trans-differentiation and for maintaining pluripotency of stem cells. In this study, we investigated the possibility of replacing all reprogramming viral factors with small molecules. To this end, we evaluated the effects of carcinogens at nongenotoxic levels on somatic cells. We identified 16 candidate chemicals through biology-oriented in silico high-throughput screening with commercially available inventories from Sigma-Aldrich for cancer research, and established a reprogramming protocol of 16-day treatment followed by 5 days of recovery. This protocol was applied to B6/129 mouse embryonic fibroblasts (MEFs) at passage 3. From recovery day 2, colonies appeared at an efficiency of 0.02%. These colonies were positive for both alkaline phosphatase and surface specific embryonic antigen-1 (SSEA-1) at a comparable level to those of mouse embryonic stem cells (ESCs). Global gene expression analysis with a 38K gene MEEBO microarray revealed that the colonies had 564 (1.5%) differentially expressed genes compared to MEFs at day 0 of treatment, and these genes were enriched in "neuromuscular differentiation." Moreover, 122 differentially expressed genes in the colonies were ESC-enriched, including downregulated somatic markers and upregulated stem cell markers. In conclusion, combined chemical treatment of MEFs herein might have caused these cells to transverse to an intermediate state within the mesodermal lineages.

Developmental Plasticity Revealed by Lineage Switch from Committed Megakaryocytic Cells to Granulocytic Cells.

Several lines of evidence showing lineage transdifferentiation within the hematopoietic system support hematopoietic plasticity. However, such lineage transdifferentiation typically requires overexpression of transcription factors or drug treatment. Here, we report that a switch from the megakaryocytic (Mk) lineage to the granuloyctic (G) lineage can be achieved by simply modifying the in vitro cytokine combination. Mk cultures were initiated with human CD34+ mobilized peripheral blood progenitor cells in serum-free medium supplemented with thrombopoietin (Tpo). CD41highCD15− cells were selected by MoFlo sorting on Day 5 (MkG5), Day 7 (MkG7) and Day 10 (MkG10). Prior to sorting, CD41+ Mks were 10%, 20%, and 25–30% polyploid (≥8N) at Day 5, 7, and 10 respectively. Sorted Mks (>97% CD41highCD15− with the remainder CD41−CD15−) were recultured in serum-containing medium with G-CSF, IL-3, IL-6 and SCF, a cytokine cocktail normally used to induce granulocytic differentiation of CD34+ progenitors (G medium). Cell numbers remained constant or decreased by as much as 30% during the first 7 days, and then increased. The MkG7 culture expanded 76-fold during the next 7 days of reculturing. By contrast, the MkG5 and MkG10 cultures expanded only 5-fold and 4-fold, respectively. After five days of reculturing, some of the MkG7 cells already showed G morphology - band-shaped nuclei - reminiscent of the fourth, or metamyelocyte, stage of granulopoiesis. Other cells, however, still showed the morphology of large polypoid Mk cells. After 7 days of reculturing, co-expression of CD41 and CD15 was seen on most cells, as assessed by immunofluorescence microscopy. However, cells started to lose CD41 expression thereafter. Only 10–20% of the cells were still CD41+ after 11 days and no CD41 expression was detected by immunofluorescence after 14 days. More than 90% of the cells expressed CD15 after 14 days of reculturing as measured by flow cytometry (Figure: right). Furthermore, more than 40% of the cells displayed nuclear segmentation, the hallmark of mature granulocytes (Figure: left). The MkG5 and MkG10 cultures also exhibited transdifferentiation of megakaryocytes into granulocytes. Surprisingly, Mks sorted at a later developmental stage exhibited more extensive G differentiation. After 14 days of reculturing, they exhibited a higher level of maturation as evidenced by flow cytometry. Additionally, the more mature Mk cells transdifferentiated at a faster rate as indicated by Giemsa staining. Large numbers of segmented granulocytes were first detected at day 11 after sorting of the MkG10 culture (25% segmented), day 14 of the MkG7 culture (40–50%) and day 17 of the MkG5 culture (10–20%). Compared to CD34+ cell-derived granulocytes cultured for the same length of time in G medium, the MkG7 and MkG10 cultures contained a greater fraction of mature (CD11b+) granulocytes. In conclusion, our data convincingly demonstrate the transdifferentiation potential of committed and mature megakaryocytes. [Display omitted]