Factor VIIa Regulates the Level of Cell-Surface Tissue Factor through Separate but Cooperative Mechanisms

Procoagulant activity of tissue factor (TF) in response to injury or inflammation is accompanied with cellular signals which determine the fate of cells. However, to prevent excessive signalling, TF is rapidly dissipated through release into microvesicles, and/or endocytosis. To elucidate the mechanism by which TF signalling may become moderated on the surface of cells, the associations of TF, fVII/fVIIa, PAR2 and caveolin-1 on MDA-MB-231, BxPC-3 and 786-O cells were examined and compared to that in cells lacking either fVII/fVIIa or TF. Furthermore, the localisation of labelled-recombinant TF with cholesterol-rich lipid rafts was explored on the surface of primary human blood dermal endothelial cells (HDBEC). Finally, by disrupting the caveolae on the surface of HDBEC, the outcome on TF-mediated signalling was examined. The association between TF and PAR2 was found to be dependent on the presence of fVIIa. Interestingly, the presence of TF was not pre-requisite for the association between fVII/fVIIa and PAR2 but was significantly enhanced by TF, which was also essential for the proliferative signal. Supplementation of HDBEC with exogenous TF resulted in early release of fVII/fVIIa from caveolae, followed by re-sequestration of TF-fVIIa. Addition of labelled-TF resulted in the accumulation within caveolin-1-containing cholesterol-rich regions and was also accompanied with the increased assimilation of cell-surface fVIIa. Disruption of the caveolae/rafts in HDBEC using MβCD enhanced the TF-mediated cellular signalling. Our data supports a hypothesis that cells respond to the exposure to TF by moderating the signalling activities as well as the procoagulant activity of TF, through incorporation into the caveolae/lipid rafts.

Tumor suppressor function of Gata2 in Acute Promyelocytic Leukemia

Most patients with acute promyelocytic leukemia (APL) can be cured with combined All Trans Retinoic Acid (ATRA) and Arsenic Trioxide therapy, which induce the destruction of PML-RARA, the initiating fusion protein for this disease1. However, the underlying mechanisms by which PML-RARA initiates and maintains APL cells are still not clear. We therefore identified genes that are dysregulated by PML-RARA in mouse and human APL cells, and prioritized GATA2 for functional studies because 1) it is highly expressed in pre-leukemic cells expressing PML-RARA, 2) its high expression persists in transformed APL cells, and 3) spontaneous somatic mutations of GATA2 occur during APL progression in both mice and humans. These and other findings suggested that GATA2 may be upregulated to thwart the proliferative signal generated by PML-RARA, and that its inactivation by mutation (and/or epigenetic silencing) may accelerate disease progression in APL and other forms of AML. Indeed, biallelic knockout of Gata2 with CRISPR/Cas9-mediated gene editing increased the serial replating efficiency of PML-RARA-expressing myeloid progenitors (and also progenitors expressing RUNX1-RUNX1T1, or deficient for Cebpa), increased mouse APL penetrance, and decreased latency. Restoration of Gata2 expression suppressed PML-RARA-driven aberrant self-renewal and leukemogenesis. Conversely, addback of a mutant GATA2R362G protein associated with APL and AML minimally suppressed PML-RARA-induced aberrant self-renewal, suggesting that it is a loss-of-function mutation. These studies reveal a potential role for Gata2 as a tumor suppressor in AML, and suggest that restoration of its function (when inactivated) may provide benefit for AML patients.

Low JAK2 V617F Allele Burden in Ph-Negative Chronic Myeloproliferative Neoplasms Is Associated with Additional CALR or MPL Gene Mutations

JAK2 (Janus kinase 2) V617F, CALR (Calreticulin) exon 9, and MPL (receptor for thrombopoietin) exon 10 mutations are associated with the vast majority of Ph-negative chronic myeloproliferative neoplasms (MPNs). These mutations affect sequential stages of proliferative signal transduction and therefore, after the emergence of one type of mutation, other types should not have any selective advantages for clonal expansion. However, simultaneous findings of these mutations have been reported by different investigators in up to 10% of MPN cases. Our study includes DNA samples from 1958 patients with clinical evidence of MPN, admitted to the National Research Center for Hematology for genetic analysis between 2016 and 2019. In 315 of 1402 cases (22.6%), CALR mutations were detected. In 23 of these 315 cases (7.3%), the JAK2 V617F mutation was found in addition to the CALR mutation. In 16 from 24 (69.6%) cases, with combined CALR and JAK2 mutations, V617F allele burden was lower than 1%. A combination of JAK2 V617F with MPL W515L/K was also observed in 1 out of 1348 cases, only. JAK2 allele burden in this case was also lower than 1%. Additional mutations may coexist over the low background of JAK2 V617F allele. Therefore, in cases of detecting MPNs with a low allelic load JAK2 V617F, it may be advisable to search for other molecular markers, primarily mutations in exon 9 of CALR. The load of the combined mutations measured at different time points may indicate that, at least in some cases, these mutations could be represented by different clones of malignant cells.

Single-molecule functional anatomy of endogenous HER2-HER3 heterodimers

Human epidermal growth factor receptors (HERs) are the primary targets of many directed cancer therapies. However, the reason a specific dimer of HERs generates a stronger proliferative signal than other permutations remains unclear. Here, we used single-molecule immunoprecipitation to develop a biochemical assay for endogenously-formed, entire HER2-HER3 heterodimers. We observed unexpected, large conformational fluctuations in juxta-membrane and kinase domains of the HER2-HER3 heterodimer. Nevertheless, the individual HER2-HER3 heterodimers catalyze tyrosine phosphorylation at an unusually high rate, while simultaneously interacting with multiple copies of downstream signaling effectors. Our results suggest that the high catalytic rate and multi-tasking capability make a concerted contribution to the strong signaling potency of the HER2-HER3 heterodimers.

The hair cycle underlies regulation of Langerhans cell renewal

ABSTRACTIn the epidermis, Langerhans cells (LCs) provide an essential link between the innate and adaptive immune systems. They self-renew in situ and continuously transport antigen from skin to lymph node (LN) T cells in the steady state. The cyclic renewal of hair follicles (HF) causes profound alterations in the cutaneous microenvironment, however little is known about its impact on LC homeostasis. Here we show that mouse LCs developed normally in the absence of hair but perceived critical transition periods in the hair cycle. LCs underwent a proliferation burst during the HF growth phase (anagen). Reinitiation or abolishment of anagen as well as loss of the HF had direct consequences on LC self-renewal. Because dividing LCs were found close to the anagen HF, we searched for the proliferative signal within this structure and identified increased Il34 expression by HF stem cells and their progeny. Inhibition of the IL-34 receptor CSF-1R at the onset of anagen completely and specifically blocked LC proliferation. Altogether, our findings demonstrate that the hair cycle directly oversees LC self-renewal and migration.

Fumarate Mediates a Chronic Proliferative Signal in Fumarate Hydratase-Inactivated Cancer Cells by Increasing Transcription and Translation of Ferritin Genes

ABSTRACT Germ line mutations of the gene encoding the tricarboxylic acid (TCA) cycle enzyme fumarate hydratase (FH) cause a hereditary cancer syndrome known as hereditary leiomyomatosis and renal cell cancer (HLRCC). HLRCC-associated tumors harbor biallelic FH inactivation that results in the accumulation of the TCA cycle metabolite fumarate. Although it is known that fumarate accumulation can alter cellular signaling, if and how fumarate confers a growth advantage remain unclear. Here we show that fumarate accumulation confers a chronic proliferative signal by disrupting cellular iron signaling. Specifically, fumarate covalently modifies cysteine residues on iron regulatory protein 2 (IRP2), rendering it unable to repress ferritin mRNA translation. Simultaneously, fumarate increases ferritin gene transcription by activating the NRF2 (nuclear factor [erythroid-derived 2]-like 2) transcription factor. In turn, increased ferritin protein levels promote the expression of the promitotic transcription factor FOXM1 (Forkhead box protein M1). Consistently, clinical HLRCC tissues showed increased expression levels of both FOXM1 and its proliferation-associated target genes. This finding demonstrates how FH inactivation can endow cells with a growth advantage.

Abstract 142: Role of CK2α in Heart Tube Proliferation

Congenital heart disease (CHD) groups a number of structural malformations of the heart and associated blood vessels present at birth. CHD is the most common birth defect, arise from defective cardiogenesis, and can be due to genetic and/or environmental factors that affect cellular processes such as proliferation during heart formation. The highly conserved serine-threonine kinase, CK2α, has been implicated in proliferative processes, including cancer, and it is a target of environmental toxins. To test whether CK2α plays a role in cardiogenesis, we used a CK2α deficient mouse. CK2α-/- mice die around embryonic day (E)11 and showed dysmorphic outflow tracts (OFT), hypoplastic right ventricles (RV) and poorly developed ventricular trabeculations. These defects, the probable cause of the lethality, could be due to deficient proliferation. Thus, we tested whether reduced CK2α expression leads to proliferative signal defects during cardiogenesis. We used three or more pairs of somite-matched wild-type and CK2α-/- embryos for the analysis of proteins by immunofluorescence and immunoblot, and of transcript expression by RT-qPCR and in situ hybridization. At E9.5 and E10.5, the number of Isl1+ cells in the OFT and the levels of Isl1 transcript in the heart of CK2α-/- embryos was no different from wild-type. These data suggest that the mechanism underlying the OFT and RV defects is different from progenitor incorporation defects. CK2α deficient hearts expressed pan-cardiac gene and protein markers normally at E9.5 and E10.5; and apoptosis was not affected, indicating that the heart malformations may be due to proliferative defects. Indeed, mitotic rates were decreased throughout the heart in CK2α mutants compared to wild-type embryos at E9.5 and E10.5; but a mitotic defect was not apparent at E8.5. The observed decrease in proliferation correlated with decreased expression of cardiac proliferative markers such as n-myc and decrease in proliferative signal pathways in the heart tube. In conclusion, these data show that, from E9.5 on, CK2α is necessary for proliferation and proliferative signal pathway activation in the heart tube; and that cellular mechanisms other than progenitor incorporation may play a key role in OFT and RV defects.