Lucie Lanikova

Differential Sensitivity to JAK Inhibitory Drugs by Isogenic Human Erythroblasts and Hematopoietic Progenitors Generated from Patient‐Specific Induced Pluripotent Stem Cells

Disease‐specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease‐causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2‐wild‐type and four lines homozygous for JAK2‐V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient‐derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild‐type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2‐V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self‐renewal of CD34+ hematopoietic progenitors. The iPSC‐mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis. Stem Cells 2014;32:269–278

The homozygous VHL(D126N) missense mutation is associated with dramatically elevated erythropoietin levels, consequent polycythemia, and early onset severe pulmonary hypertension.

von Hippel‐Lindau (VHL) protein is the principal negative regulator of hypoxia sensing mediated by transcription factors. Mutations in exon 3 of the VHL gene lead to Chuvash (VHLR200W) and Croatian (VHLH191D) polycythemias. Here, we describe an infant of Bangladesh ethnicity with a novel homozygous VHLD126N mutation with congenital polycythemia and dramatically elevated erythropoietin (EPO) levels, who developed severe fatal pulmonary hypertension. In contrast to Chuvash polycythemia, erythroid progenitors (BFU‐Es) did not reveal a marked EPO hypersensitivity. Further, NF‐E2 and RUNX1 transcripts that correlate with BFU‐Es EPO hypersensitivity in polycythemic mutations were not elevated. Pediatr Blood Cancer 2014;61:2104–2106.

RUNX1 and NF-E2 upregulation is not specific for MPNs but is seen in polycythemic disorders with augmented HIF signaling

Overexpression of transcription factors runt-related transcription factor 1 (RUNX1) and nuclear factor, erythroid-derived 2 (NF-E2) was reported in granulocytes of patients with polycythemia vera and other myeloproliferative neoplasms (MPNs). Further, a transgenic mouse overexpressing the NF-E2 transgene was reported to be a model of MPN. We hypothesized that increased transcripts of RUNX1 and NF-E2 might characterize other polycythemic states with primary polycythemic features, that is, those with exaggerated erythropoiesis due to augmented erythropoietin (EPO) sensitivity. We tested the expression of RUNX1 and NF-E2 in polycythemic patients of diverse phenotypes and molecular causes. We report that RUNX1 and NF-E2 overexpression is not specific for MPN; these transcripts were also significantly elevated in polycythemias with augmented hypoxia-inducible factor activity whose erythroid progenitors were hypersensitive to EPO. RUNX1 and NF-E2 overexpression was not detected in patients with EPO receptor (EPOR) gain-of-function, suggesting distinct mechanisms by which erythroid progenitors in polycythemias with defects of hypoxia sensing and EPOR mutations exert their EPO hypersensitivity.

β‐Thalassemia Due to Intronic LINE‐1 Insertion in the β‐Globin Gene (HBB): Molecular Mechanisms Underlying Reduced Transcript Levels of the β‐GlobinL1 Allele

We describe the molecular etiology of β+‐thalassemia that is caused by the insertion of the full‐length transposable element LINE‐1 (L1) into the intron‐2 of the β‐globin gene (HBB). The transcript level of the affected β‐globin gene was severely reduced. The remaining transcripts consisted of full‐length, correctly processed β‐globin mRNA and a minute amount of three aberrantly spliced transcripts with a decreased half‐life due to activation of the nonsense‐mediated decay pathway. The lower steady‐state amount of mRNA produced by the β‐globinL1 allele also resulted from a reduced rate of transcription and decreased production of full‐length β‐globin primary transcripts. The promoter and enhancer sequences of the β‐globinL1 allele were hypermethylated; however, treatment with a demethylating agent did not restore the impaired transcription. A histone deacetylase inhibitor partially reactivated the β‐globinL1 transcription despite permanent β‐globinL1 promoter CpG methylation. This result indicates that the decreased rate of transcription from the β‐globinL1 allele is associated with an altered chromatin structure. Therefore, the molecular defect caused by intronic L1 insertion in the β‐globin gene represents a novel etiology of β‐thalassemia.

Evolutionary selected Tibetan variants of HIF pathway and risk of lung cancer

Tibetans existed in high altitude for ~25 thousand years and have evolutionary selected unique haplotypes assumed to be beneficial to hypoxic adaptation. EGLN1/PHD2 and EPAS1/HIF-2α, both crucial components of hypoxia sensing, are the two best-established loci contributing to high altitude adaptation. The co-adapted Tibetan-specific haplotype encoding for PHD2:p.[D4E/C127S] promotes increased HIF degradation under hypoxic conditions. The Tibetan-specific 200 kb EPAS1 haplotype introgressed from an archaic human population related to Denisovans which underwent evolutionary decay; however, the functional variant(s) responsible for high-altitude adaptation at EPAS1/HIF-2α have not yet been identified. Since HIF modulates the behavior of cancer cells, we hypothesized that these Tibetan selected genomic variants may modify cancer risk predisposition. Here, we ascertained the frequencies of EGLN1D4E/C127S and EGLN1C127S variants and ten EPAS1/HIF-2α variants in lung cancer patients and controls in Nepal, whose population consists of people with Indo-Aryan origin and Tibetan-related Mongoloid origin. We observed a significant association between the selected Tibetan EGLN1/PHD2 haplotype and lung cancer (p=0.0012 for D4E, p=0.0002 for C127S), corresponding to a two-fold increase in lung cancer risk. We also observed a two-fold or greater increased risk for two of the ten EPAS1/HIF-2α variants, although the association was not significant after correcting for multiple comparisons (p=0.12). Although these data cannot address the role of these genetic variants on lung cancer initiation or progression, we conclude that some selected Tibetan variants are strongly associated with a modified risk of lung cancer.

Loss of Major DNase I Hypersensitive Sites in Duplicated β-globin Gene Cluster Incompletely Silences HBB Gene Expression

We report an infant with sickle cell disease phenotype by biochemical analysis whose β‐globin gene (HBB) sequencing showed sickle cell mutation (HBBS) heterozygosity. The proband has a unique head‐to‐tail duplication of the β‐globin gene cluster having wild‐type (HBBA) and HBBS alleles inherited from her father; constituting her HBBS/HBBS‐HBBA genotype. Further analyses revealed that probands duplicated β‐globin gene cluster (∼650 kb) encompassing HBBA does not include the immediate upstream locus control region (LCR) or 3′ DNase I hypersensitivity (HS) element. The LCR interacts with β‐globin gene cluster involving long range DNA interactions mediated by various transcription factors to drive the regulation of globin genes expression. However, a low level of HBBA transcript was clearly detected by digital PCR. In this patient, the observed transcription from the duplicated, distally displaced HBBA cluster demonstrates that the loss of LCR and flanking 3′HS sites do not lead to complete silencing of HBB transcription.

Wnt Effector TCF4 Is Dispensable for Wnt Signaling in Human Cancer Cells

T-cell factor 4 (TCF4), together with β-catenin coactivator, functions as the major transcriptional mediator of the canonical wingless/integrated (Wnt) signaling pathway in the intestinal epithelium. The pathway activity is essential for both intestinal homeostasis and tumorigenesis. To date, several mouse models and cellular systems have been used to analyze TCF4 function. However, some findings were conflicting, especially those that were related to the defects observed in the mouse gastrointestinal tract after Tcf4 gene deletion, or to a potential tumor suppressive role of the gene in intestinal cancer cells or tumors. Here, we present the results obtained using a newly generated conditional Tcf4 allele that allows inactivation of all potential Tcf4 isoforms in the mouse tissue or small intestinal and colon organoids. We also employed the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system to disrupt the TCF4 gene in human cells. We showed that in adult mice, epithelial expression of Tcf4 is indispensable for cell proliferation and tumor initiation. However, in human cells, the TCF4 role is redundant with the related T-cell factor 1 (TCF1) and lymphoid enhancer-binding factor 1 (LEF1) transcription factors.